Poly[oxy(methyl-1,2-ethanediyl)], α-hydro-ω-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), 2-propenoate

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:

From July 18, 2016 to August 23, 2016

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

DOC measurement using a TOC analyser

Details on sampling:

For the experiment, the water-accommodated fractions (WAF) of the concentrations to be tested were prepared. This was done by weighing the nominal load (real loadings: 1.7 mg/L, 3.3 mg/L, 10.4 mg/L, 32.4 mg/L and 100.7 mg/L), adding the appropriate amount of algal medium (demineralised water enriched with minerals but without algae) and shaking for 25 hours. After membrane filtration, the solutions were used to prepare the treatments.

Vehicle:

no

Details on test solutions:

At the start and at the end of the test of the experiment, the content of the test substance in the test solutions was determined using DOC measurement. The measured concentrations lay between 31 % and 88 % of the nominal concentration at the beginning of the test and between 37% and 159% of the nominal concentration at the end of the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture 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 was prepared.
Four days before the start of each test, an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours. The resulting culture grew exponentially. Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.0 – 22.2 °C

pH:

7.5-8.9

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Treatments tested: 1.0, 3.2, 10, 32 and 100 mg/L (nominal concentrations)

Details on test conditions:

Number of replicates: 6 replicates for the control and 3 replicates for each treatment
Vessels: glass flasks total volume 65 mL
Lighting: 5300 Lux
- Blank control: deionised water with nutrient medium and algae
- Treatments: test solution and algae

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7) (CAS No. 7778-50-9)

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

<= 1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

<= 1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 1.2 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.:

2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

4 experiments were performed. In the 1st and 2nd experiment one of the validity criteria was not met. Therefore, the experiment was repeated. A 3rd experiment was performed like the first two experiments. It was performed using 5 concentrations ranging from 1.0 to 100 mg/L. The water-accommodated fraction (WAF) of each single concentration to be tested was prepared. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times. Statistical significant inhibition of algal growth was observed in all tested concentrations. Therefore, a 4th experiment was performed, using 7 concentrations ranging from 0.1 to 100 mg/L. The water-accommodated fraction (WAF) of the highest concentration (100 mg/L nominal) was prepared. The lower treatments were prepared by dilution of this stock WAF (according to OECD Guidance Document No. 23 were is stated that serial dilution of a stock WAF may be the only applicable method for chemicals being toxicshowing algal growth inhibition at concentrations of 1 mg/L or lower). Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times. Again, inhibition of algal growth was observed in all tested concentrations. Therefore – as a worst-case scenario – the 3rd experiment using preparation of each WAF individually was chosen for determination of the biological results and EC values, NOEC and LOEC were based on the data of the 3rd experiment.

Results with reference substance (positive control):

The 72h-EC50s of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

Table 1. Endpoints

Endpoints	NOEC	LOEC	EC10	EC50
Growth rate	<1.2 mg/mL	≤ 1.2 mg/mL	2.1 mg/L	15 mg/L
Yield	<1.2 mg/mL	≤ 1.2 mg/mL	0.38 mg/L	2.7 mg/L

- Normal and healthy appearance of the algae: yes

Analytical Determination:

At the start and at the end of the test of the experiment, the content of the test substance in the test solutions was determined using DOC measurement. The measured concentrations lay between 31 % and 88 % of the nominal concentration at the beginning of the test and between 37% and 159% of the nominal concentration at the end of the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations. Geometric mean was calculated by multiplication of the n participating concentrations and taking the n^throot.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72h ErC50 (growth rate) and EyC50 (yield inhibition) for Desmodesmus subspicatus based on measured concentrations were determined to be 15.0 and 2.7 mg/L, respectively, while the NOEC for both the parameters was <1.2 mg/L. Further, the 72h ErC10 and EbC10 were determined to be at 2.1 and 0.38 mg/L, respectively.

Executive summary:

A study was conducted to determine the toxicity to aquatic algae and cyanobacteria of the test substance according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Algae were exposed to Water Accommodated Fractions (WAF; using an orbital shaker) of the substance at loading rates of 0, 1, 3.2, 10, 32 and 100 mg/L (nominal concentrations) and incubated in open (covered with perforated plastic foil) for 72 h in triplicate. The cell concentration of each replicate was determined by measuring the cell numbers every 24 h with an electronic particle counter. Growth rate and the yield were determined from the cell number at the respective observation times (i.e., at 0, 24, 48 and 72 h). Samples were collected for analytical dose verification at the start and at the end of the test. Analysis of the samples indicated measured concentrations between 31 and 88% of the nominal concentration at the beginning of the test and between 37 and 159% of the nominal concentration at the end of the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations. The 72 h EC50 of positive control (potassium dichromate) within the range of the laboratory standards (growth rate: 0.73 - 1.10 mg/L; yield: 0.21–0.66 mg/L). All validity criteria were met. Under the study conditions, the 72 h ErC50 (growth rate) and EyC50 (yield inhibition) forDesmodesmus subspicatusbased on measured concentrations were determined to be 15.0 and 2.7 mg/L, respectively, while the NOEC for both the parameters was <1.2 mg/L. Further, the 72 h ErC10 and EbC10 were determined to be at 2.1 and 0.38 mg/L, respectively (Bangert, 2016).

Key value for chemical safety assessment

EC50 for freshwater algae:

15 mg/L

EC10 or NOEC for freshwater algae:

2.1 mg/L

Additional information

A study was conducted to determine the toxicity to aquatic algae and cyanobacteria of the test substance according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Algae were exposed to Water Accommodated Fractions (WAF; using an orbital shaker) of the substance at loading rates of 0, 1, 3.2, 10, 32 and 100 mg/L (nominal concentrations) and incubated in open (covered with perforated plastic foil) for 72 h in triplicate. The cell concentration of each replicate was determined by measuring the cell numbers every 24 h with an electronic particle counter. Growth rate and the yield were determined from the cell number at the respective observation times (i.e., at 0, 24, 48 and 72 h). Samples were collected for analytical dose verification at the start and at the end of the test. Analysis of the samples indicated measured concentrations between 31 and 88% of the nominal concentration at the beginning of the test and between 37 and 159% of the nominal concentration at the end of the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations. The 72 h EC50 of positive control (potassium dichromate) within the range of the laboratory standards (growth rate: 0.73 - 1.10 mg/L; yield: 0.21–0.66 mg/L). All validity criteria were met. Under the study conditions, the 72 h ErC50 (growth rate) and EyC50 (yield inhibition) forDesmodesmus subspicatusbased on measured concentrations were determined to be 15.0 and 2.7 mg/L, respectively, while the NOEC for both the parameters was <1.2 mg/L. Further, the 72 h ErC10 and EbC10 were determined to be at 2.1 and 0.38 mg/L, respectively (Bangert, 2016).