Glycerol, propoxylated, esters with acrylic acid, reaction products with diethylamine

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:

2017-02-03 to

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the request of the Sponsor’s representative, no analysis was performed after 72 hours of exposure (it is known that the test item is unstable in aqueous media and thus analysis after end of exposure was not relevant: samples were taken from all test concentrations at the start of the test then analysed according to the analytical procedure.

Vehicle:

no

Details on test solutions:

Tests solutions were prepared from dilutions of a stock solution at 1 g/L of nominal concentration: a known volume of test item (196.1 µg/L) was poured into a volumetric flask, the volume was then made up to 200 mL with test medium. The solution was kept under high speed stirring at ambient temperature during approximately 1 hour with a magnetic stir bar. No filtration was necessary before dilution.

Test organisms (species):

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

Details on test organisms:

The test organism used for the study was Pseudokirchneriella subcapitata, Strain No. CCAP 278/4, supplied by the Culture Centre of Algae and Protozoa (Ambleside, UK).
Transfers of P. subcapitata were made regularly to provide suitable subcultures. The algae were cultivated under standardized conditions as described in Annex 2 of the OECD 201 guideline.
The quality of the stock culture was checked for the absence of micro-organisms and deformed or abnormal cells under microscopic observation before use.
Four days before the start of the exposure, two pre-cultures were prepared by inoculating sufficient cells from the algal stock culture to the growth medium to obtain a low cell density, e.g. 2.103 cells/mL to 104 cells/mL for pre-culturing, in order to maintain exponential growth until the start of the test. The pre-cultures were incubated under the same conditions as those used for the test cultures. Only one of the two pre-cultures was used to inoculate the test flasks for the study. The second one was only to be used if the first one was damaged.
At the beginning of the test, the cell density of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to obtain an initial cell concentration of 5.103 cells/mL as recommended in the OECD 201 guideline.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 23 ± 1°C: range from 22.8 to 23.1°C (mean: 22.9°C).

pH:

0h: 8.2 - 8.8
72h: 7.9 - 8.1

Dissolved oxygen:

0h: 8.1 - 8.3 mg/L
72h: 8.8 - 9.3 mg/L

Nominal and measured concentrations:

Nominal concentrations (in mg/L): 0 (Control), 0.52, 1.32, 3.40, 8.80, 22.80, 59.60, 154.00

Details on test conditions:

The incubation was conducted in a phytoculture cabinet that allows test flasks to be incubated under precise conditions: temperature was set to 23 ± 1°C; flasks were continuously shaken with a rotation speed at around 100 rpm and constantly illuminated by fluorescent tubes between 6,000 and 10,000 lux.

Test vessels were made of glass bottles (120 mL capacity) capped with cellulose bungs. They were filled with a volume of 50 mL.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

10.99 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

54.77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, no precipitation was observed at the end of the test.

Microscopic observation confirmed that the algae appeared "normal" at the end of the test: the normal shape of P. subcapitata algae is a crescent shaped cell with an average length of 5-10 µm.

Analytical measurements: as there was evidence that the concentration of the substance being tested was determined to be within ± 20% of the nominal concentration at the start of the test, the exposure concentrations were based on the nominal concentrations.

Reference Item Test

The sensitivity of the test system and the methodology are evaluated every two months by performing an algal growth inhibition test on potassium dichromate. The latest value of ErC50 obtained in December 2016 was 1.18 mg/L.

 

According to the ISO 8692 standard, 72h-ErC50 obtained from an inter-laboratory exercise on potassium dichromate was in the range from 0.65 to 1.73 mg/L.

Algae Test

The test item had a statistically significant inhibitory effect on the growth rate and on the yield of the algae after the test period of 72 hours at 1.32 mg/L and 3.40 mg/L respectively.

Percentage of inhibition

Test solution (mg/L)	Growth rate Iµi (%)	Yield Iyi (%)
0.52	-2.0	-8.4
1.32	3.3	12.9
3.40	5.7	21.0
8.80	13.3	42.4
22.80	24.0	63.1
59.60	49.5	87.9
154.00	84.1	98.3

Validity criteria fulfilled:

yes

Conclusions:

The results are reported as growth rate and yield inhibitions. The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) and yield (72h-EyC10 and 72h-EyC50, respectively) were determined:

GROWTH RATE:
EC10 = 10.99 mg/L (95% confidence interval: 8.55 - 14.13 mg/L)
EC50 = 54.77 mg/L (95% confidence interval: 40.42 - 73.55 mg/L)

YIELD:
EC10 = 1.56 mg/L (95% confidence interval: 0.97 - 2.53 mg/L)
EC50 = 11.35 mg/L (95% confidence interval: 6.53 - 19.69 mg/L)

Executive summary:

This study was designed to determine the effects of the test item on the growth of Pseudokirchneriella subcapitata in a 72-hours test according to the OECD 201 Guideline.

 

Tests solutions were prepared from dilutions of a stock solution.

 

At the request of the Sponsor’s representative, no analysis was performed after 72 hours of exposure (it is known that the test item is unstable in aqueous media and thus analysis after end of exposure was not relevant: as there was evidence that the concentration of the substance being tested was determined within ± 20% of the nominal concentration at the start of the test, analysis of the results was based on nominal values.

 

The results are reported as growth rate and yield inhibitions. The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) and yield (72h-EyC10 and 72h-EyC50, respectively) were determined:

 

Growth Function	72h-EC10 (mg/L)	95% confidence level		72h-EC50 (mg/L)	95% confidence level
		Lower limit	Upper limit		Lower limit	Upper limit
Growth rate	10.99	8.55	14.13	54.77	40.42	73.55
Yield	1.56	0.97	2.53	11.35	6.53	19.69

The definitive test met the validity criteria of the test guideline detailed as follows:

§ The biomass in the control cultures increased exponentially by a factor of 55.8 within the 72-hour test period (required: at last 16-fold)

§ The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2 -3) in the control cultures were less than or equal to 18.4% (required: < 35%)

§ The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 2.2% (required: < 7%)

 

Description of key information

A study on the toxicity of Glycerol, propoxylated, esters with acrylic acid, reaction products with diethylamine (CAS 162492-10-6) to freshwater algae and cyanobacteria was conducted according to the OECD Test Guideline 201 (Growth Inhibition Test).

As the test item is known to be unstable in aqueous media, analytical measurements were performed only at the beginning of the exposure period. As there was evidence that the concentrations of the test substance were within +-20% of the nominal concentrations at the start of the test, the analysis of the results was based on nominal values.

The growth rate EC10 and EC50 were determined to be equal to 10.99 and 54.77mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

54.77 mg/L

EC10 or NOEC for freshwater algae:

10.99 mg/L