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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April, 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: crystalline
Details on test material:
Purity = 99.8%
Specific details on test material used for the study:
Test item identification : N,N'-Methylenediacrylamide
Batch number : LQ27A1696
Internal number : 2926879-001
Manufacturing date : January 14, 2016
Expiry date : January 13, 2017
Chemical name : N,N'-Methylenediacrylamide
C.A.S. number : 110-26-9
EEC number : 203-750-9
Molecular weight : 154.17 g/mol
Empirical formula : C7H10N2O2
Purity : > 99%

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
The test concentration was chosen in accordance with the results of pre-tests (range-finding tests), whose relevant Raw Data were archived under the code of the present study.
The main test included only one test item concentration equal to 100.0 mg test item/L, corresponding to 99.8 mg/L as a.i. (calculated considering test item purity, 99.8% w/w, as reported in Certificate of Analysis supplied by the Sponsor).
The test medium was prepared as follows:
Prior to the test start, 500 mL of a 100.0 mg test item/L stock solution was prepared by direct weighing into algal culture medium (0.0500 g of test item into 500 mL of algal culture medium).
The obtained solution showed insolubilized flakes of test item, so it was sonicated for about 5 minutes. After sonication, it was clear and transparent.
A negative control with algal growth medium only was also tested

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The study was performed with the unicellular, fresh-water green algae Pseudokirchneriella subcapitata strain (formerly known as Selenastrum capricornutum), cultured in the laboratories of the Test Facility and originally purchased from the Institute of Plant Physiology of the University of Göttingen, Germany.
The algae were cultured in a climatic chamber at 24 °C ± 2 °C under continuous uniform illumination in the range 4440 - 8880 Lux in the spectral range 400-700 nm. The culture medium was the same of the test medium; the stock cultures were weekly transferred to fresh medium and maintained in continuous shaking to ensure the necessary amount of CO2and to keep algae in suspension. Cultures containing deformed or abnormal cells were discarded. Only exponentially growing algal cultures have been used to start the test.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h

Test conditions

Test temperature:
24.0 – 24.1 °C.
7.82 – 7.96 at the test start.
9.14 – 9.23 at the test end.
Nominal and measured concentrations:
In fresh solution the analytical recovery was 97.9% of the nominal concentration (100 mg/L).
In spent solution (after 72 hours of exposure) the analytical recovery of the concentration was 102.0% of the nominal value.
Since the analytical recoveries of N,N'-Methylenediacrylamide were in the range 80 % - 120 %, the toxic end-points were calculated in terms of nominal test item concentrations (100 mg/L).
Details on test conditions:
The treatment vessels were identified by experiment number, group and replicate number.
All the described procedures were conducted under a laminar flow bench to ensure the sterility of the algal cultures.
For the negative control and for the test concentrations, 100 mL capacity conical glass flasks capped with air permeable stoppers were used.
Six replicates for the test item concentration and six for negative control were prepared. At test start, 50 mL of test solutions were poured into the test flasks and inoculated with algae to obtain a starting cell density of about 104 cells/mL.
The algal inoculum was taken from a pre-culture, started 3 days before the beginning of the test in order to ensure that the assay was performed with exponentially growing algal population. The cell density of the inoculum was around 106 cells/mL.
The test flasks were incubated in a climatic chamber, under continuous illumination and constant shaking. The flasks were randomly distributed under the light and were replaced every day during the test.
The incubation temperature was continuously monitored during the course of the study by a PT100 probe installed in the environmental test chamber. It was between 24.0 – 24.1°C during the exposure period with a standard deviation of 0.02°C. The temperature range provided by OECD guideline is 24 ± 2 C. The temperature values during the test were within the provided range.
Light conditions:
Continuous illumination; light intensity was ranged between 6098 and 6250 Lux, within the OECD recommended range (4440 - 8880 Lux).
Test duration: 72 hours
Test medium:
Reconstituted water according to OECD Guideline 201.
Test medium pH was measured with a portable pH meter at the beginning and at the end of the test. For fresh solutions, the pH values were measured sampling an aliquot immediately after test solution preparation, before pouring it in the test flasks. For spent solutions (after 72 hours), pH were measured in each test flask.
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
Key result
72 h
Dose descriptor:
Effect conc.:
100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 d
Dose descriptor:
Effect conc.:
100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:

Applicant's summary and conclusion

Validity criteria fulfilled:
The toxicity of N,N'-Methylenediacrylamide was determined in a GLP guideline study on Pseudokirchneriella subcapitata. The NOEC at 72 hours was 100 mg/L (limit test).