5-diethoxymethyl-1,3-benzodioxole

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

See explanation in result section

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-diethoxymethyl-1,3-benzodioxole

EC Number:

254-956-0

EC Name:

5-diethoxymethyl-1,3-benzodioxole

Cas Number:

40527-42-2

Molecular formula:

C12H16O4

IUPAC Name:

5-(diethoxymethyl)-2H-1,3-benzodioxole

impurity 1

Reference substance name:

Piperonal

EC Number:

204-409-7

EC Name:

Piperonal

Cas Number:

120-57-0

Molecular formula:

C8H6O3

IUPAC Name:

1,3-benzodioxole-5-carbaldehyde

Specific details on test material used for the study:

Batch no.: 19020094
Appearance: colorless to pale yellow liquid
Purity: 99.80 % (GC)
Homogeneity: homogeneous
Production date: 20. Feb. 2019
Expiry date: 20. Feb. 2019
Storage: Room Temperature (20 ± 5 °C) store under inert gas in the dark

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

* Preparations
A stock solution will be prepared by mixing the nominal load of 100 mg/L (±10 %) with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 24 ± 1 hours. Based on the results of validation of the analytical method, after this, complete dissolution of the test item is reached. The concentrations to be tested will be prepared by dilution of this stock solution with algal medium.

* The study was performed using 5 concentrations ranging from 0.32 to 32 mg/L (nominal).

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

* Origin and Culture
The culture of Desmodesmus subspicatus was obtained from MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock cul- ture on solid agar at 2 – 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture will be prepared.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.0 to 24.7°C

pH:

7.7 to 7.8

Nominal and measured concentrations:

Treatments tested: 32 / 10 / 3.2 / 1 / 0.32 mg/L nominal concentration
The concentrations to be tested are based on non GLP pre-tests.

To avoid any loss of test item by absorption onto the algae cells, additional test replicates without algae were prepared only for analytical measurement.
At the beginning of the test and after 24 hours, the correlation between nominal and measured concentration was very good (range 90 % and 107 % of the nominal concentrations).
After 72 hours the measured concentrations were in a range of 33 % - 97 % of the nominal concentration.
Because of insufficient stability under test conditions, after 72 hours only very low and strong scattering test item concentration were measurable.

Details on test conditions:

For each treatment, 200 mL of the respective test item solution was mixed with the necessary amount of algal pre-culture (0.389 mL) to achieve a cell concentration of 3 *10^3 cells/mL. For the blank control, 350 mL nutrient medium was used instead of test item solution and mixed with the necessary amount of algal pre-culture (0.681 mL). In this mixture, the pH-value was measured. The real cell concentration at the beginning of the test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.

The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an orbital shaker to keep the algae in suspension. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter. After the test, the pH value in treatments and blank control was measured again.

At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).
The content of the test item in the test solution was analytically determined at the beginning and every 24 hours. To avoid any loss of test item by absorption onto the algae cells, analytical measurement was performed in in additional test replicates without algae.


Number of replicates: 6 replicates for the blank control; 3 replicates for each treatment
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Lighting: within the specified range (4440 – 8880 Lux)
Replicates (Blank control): 45 ±1 mL algal medium and algae; 1 vessel filled with 430 mL test solution without algae (for analytical determination)
Replicates (Treatments): 45 ±1 mL test solution and algae; 1 vessel filled with 430 mL control solution without algae (for analytical determination)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) was tested as positive control in a current reference study.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1.62 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1.62 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 2.64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 3.64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 3.13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 7.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 3.88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Any other information on results incl. tables

* Biological Results of the Reference Study (201809R301)

The results of the last study with the positive control K₂Cr₂O₇are presented in the following table.The study was performed under GLP conditions in June 2018.

Parameter	Value	95% confidence interval
72h ErC50	0.65 mg/L	0.60 mg/L – 0.71 mg/L
72h EyC50	0.28 mg/L	0.21 mg/L – 0.38 mg/L

* Deviations

a) Deviations from the Study Plan

The following deviations were observed:

- The temperature in the experiment was in the range 22.0– 24.7°C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.

- The temperature during pre-culture incubation was in the range 23.2 – 24.5°C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical. 

The deviations were assessed and signed by the study director on 27. May 2019.

b) Deviations from the Guideline         

The following deviations were observed:

- The stock solutions to be autoclaved for 20 minutes instead of the required 15 minutes. This can be considered uncritical, as the goal of sterility is achieved.       

- The temperature in the experiment was in the range 22.0– 24.7 °C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.

- The temperature during pre-culture incubation was in the range 23.2 – 24.5 °C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.

The deviations were assessed and signed by the study director on 27. May2019

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

According to OECD 201, the study was performed using 5 concentrations ranging from 0.32 to 32 mg/L (nominal) and significant inhibition of algal growth was observed at the 2 highest concentrations 10 mg/L and 32 mg/L.
The EC50-72h for Growth Rate and Yield endpoint for the test item were 3.88 mg/L and >7.02 mg/L, respectively.