1,1'-(chlorophenylmethylene)bis[4-methoxybenzene]

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:

2017-11-09 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:

adopted March 2006, corrected 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: 4,4'-Dimethoxytrityl chloride 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 4,4'-Dimethoxytrityl chloride 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 444.2 mg test item (including a factor of 1.11 to take into account the dilution caused by addition of the algal inoculum) to 1000 mL test medium and shaking for 48 h using an overhead shaker at 23.9 – 24.8 °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 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 Na2EDTA×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: in the highest test item loading rate (400 mg/L), some algal cells were thickened. However, the morphology of the algal cells in the lower test item loading rates as well as in the control showed no obvious abnormality (0, 25, 50, 100, 200 mg/L).

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:

7.3 - 7.8 (control); 6.3 - 7.6 (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, 0.194, 0.289, 0.537, 0.947, 1.8

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: constant lighting
- 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 -0.5, 39.2 and 73.3 % for yield and -3.1 %, 8.7 % and 28.5 % 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.:

0.878 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 0.520 - 1.462 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.587 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 0.387 - 0.897 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.475 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 0.306 - 0.738 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.947 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.537 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.629 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 1.079 - 2.417 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.968 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.798 - 1.168 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.738 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.628 - 0.866 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.947 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.537 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The morphology of the algal cells in the lower test item loading rates (treatment groups A, B, C and D; 25, 50, 100, 200 mg/L) as well as in the control showed no obvious abnormality. In the highest test item loading rate (treatment group E 400 mg/L), some algal cells were thickened.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no; difference can be explained by the water solubility of the substance
- Effect concentrations exceeding solubility of substance in test medium: no

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 test item conc. [mg/L]	Inhibition of Growth rate [%]
		24 h	48 h	72 h
NC	---	---	---	---
25	0.194*	8.2	4.1	3.2
50	0.289*	-0.1	-0.6	0.6
100	0.537*	4.2	1.9	3.1
200	0.947*	16.4	19.7	19.4
400	1.80	25.1	49.8	51.7

* 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. The measured test item concentrations in the test item treatments were <LOQ - 0.46% of the nominal loading rates (see table 2). 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. 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. Measured concentrations which are < LOQ are presented in brackets. LOQ: Limit of Quantification (1 mg/L). LOD: Limit of Quantification = 0.16 mg/L.

Nominal test item loading rate [mg/L]	Sampling time [h]	Measured test item concentration [mg/L]	Geometric mean test item conc. [mg/L]
NC	0	n.d	/
	24	n.d
	48	n.d
	72	n.d
25	0	< LOQ (0.193)	< LOQ (0.194)
	24	< LOQ (0.200)
	48	< LOQ (0.189)
	72	< LOQ (0.193)
50	0	< LOQ (0.294)	< LOQ (0.289)
	24	< LOQ (0.256)
	48	< LOQ (0.299)
	72	< LOQ (0.311)
100	0	< LOQ (0.524)	< LOQ (0.537)
	24	< LOQ (0.517)
	48	< LOQ (0.519)
	72	< LOQ (0.593)
200	0	< LOQ (0.949)	< LOQ (0.947)
	24	< LOQ (0.936)
	48	< LOQ (0.955)
	72	< LOQ (0.949)
400	0	1.84	1.80
	24	1.81
	48	1.78
	72	1.77

 n.d. = not detected

CRITERIA OF VALIDITY

- The biomass (cell concentration) in the control has increased by a factor of 161.6 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 12.5 % and therefore ≤ 35 %.

- The coefficient of variation of average specific growth rate during the test period in the control replicates is 0.9 % 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 EC50 based on growth rate of the test item in Pseudokirchneriella subcapitata is is 1.629 mg/L (95 % CL 1.079 - 2.417 mg/L; measured values).

Executive summary:

In a 72-hour acute toxicity study, cultures of Pseudokirchneriella subcapitata, Strain No. 61.81 SAG were exposed to 4,4'-Dimethoxytrityl chloride at measured concentrations of 0, 0.194, 0.289, 0.537, 0.947, 1.8 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. As most 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 NOEC and EC50 values based on growth rate were 0.537 and 1.629 mg a.i./L, respectively. 

The following abnormalities were noted: The microscopic examination after 72 hours did not show any obvious abnormality, except for in the highest treatment group (400 mg/L) some algal cells were thickened.

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	0.878 (0.520 - 1.462)	1.629 (1.079 - 2.417)
EC10	0.475 (0.306 - 0.738)	0.738 (0.628 - 0.866)
LOEC	0.947	0.947
NOEC	0.537	0.537

            

 

Description of key information

In a 72-hour algae growth inhibition test cultures of Pseudokirchneriella subcapitata were exposed to 4,4'-Dimethoxytrityl chloride at measured concentrations of 0, 0.194, 0.289, 0.537, 0.947, 1.8 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: 1.629 mg/L (95 % C.L. 1.079 – 2.417 mg/L);

- 72 h NOErC: 0.537 mg/L

- 72 h LOErC: 0.947 mg/L.  

Key value for chemical safety assessment

EC50 for freshwater algae:

1.629 mg/L

EC10 or NOEC for freshwater algae:

0.537 mg/L