2-[[(butylamino)carbonyl]oxy]ethyl acrylate

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:

10 - 14 Feb 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

28 Jul 2011

Deviations:

yes

Remarks:

(1) The pH (control) increased by 0.2 (8 - 8.2). Required: max. 1.5 units. (2) Light intensity was 40 instead of the recommended 60 - 120 µE*m^-2*s^-2 but enabled optimal growth to fulfill the OECD quality criteria.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

24 Aug 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health, Bern, Switzerland (26 Jan 2015)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.100, 0.316, 1.00, 3.16, 10.0, and 31.6 mg/L (nominal). The LOQ is higher than the two lowest test concentrations (0.1 and 0.316 mg/L). This has no influence on the evaluation of the test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test concentrations were prepared by dilution of a 31.6 mg/L stock solution in algal medium. The pH of the OECD medium was adjusted before inoculation with algal culture (8.0 ± 0.2) and then the test solutions were filter sterilized (0.45 µm cellulose acetate membrane sterility syringe filter, VWR, International)
- Controls: Blank controls containing test medium and algae (6 replicates).

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green unicellular microalgae
- Source: Collection of Algal Culture, Institute of Freshwater Ecology, University of Göttingen, Germany
- Method of cultivation: 250 mL flask containing 100 mL sterile OECD medium inoculated with cell material from axenic slope cultures; Illumination: continuous (2000 - 3000 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland); Temperature: 22 ± 2 °C
- Other: The culture is re-purchased yearly to control sensitivity.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.5 ± 0.6 °C

pH:

blank: 8.0 (0 h), 8.2 (72 h)
highest concentration: 7.9 (0 h), 7.8 (72 h)

Nominal and measured concentrations:

control, 0.100, 0.316, 1.00, 3.16, 10.0, and 31.6 mg/L (nominal)
control, < LOQ,

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL all-glass flasks filled with 100 mL test medium.
- Renewal rate of test solution: No renewal.
- Initial cells density: 2 - 5E+03 cells/mL = 0.5 - 0.85 µg dry weight/mL (i.e. OD680 = ca. 0.005 units)
- Control end cells density: 0.568 units (OD680)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes (OECD medium)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Same as test.
- Intervals of water quality measurement: pH was measured at 0 and 72 h in the medium which is used for the control and in the saturated solutions.

OTHER TEST CONDITIONS
- Sterile test conditions: The test solutions were filter sterilized (0.45 µm cellulose acetate membrane sterile syringe filter, VWR, International).
- Adjustment of pH: The pH of the OECD medium was adjusted before inoculation with algal culture (pH 8.0 ± 0.2).
- Photoperiod: Continuous
- Light intensity and quality: ca. 3000 lux (40 µE*m^-2*s^-2) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland). The light intensity is maintained within ± 6% of the average light intensity over the incubation area (required: ± 15%). The present light intensity is lower than the the 60 - 120 µE*m^-2*s^-2 recommended by the OECD testing guideline 202. However, based on the laboratory experience, the two lamps enable optimal growth conditions at a light intensity of 40 mE*m^-2*s^-2 to fulfill the required quality criteria according to OECD while yielding the "fittest" algal cells.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Spectrophotometer (wavelength: 680 nm) after 0, 24, 48 and 72 h.
- Other: Aliquots of 5 mL were removed from each test flask under sterile conditions. Cell concentrations at 0 h were only determined in the control vessels.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: Yes, non-GLP.
- Test concentrations: 1, 10, and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes. In the range finding test inhibition of the average growth rate was 19, 60, and 102% at concentrations 1, 10, and 100 mg/L (nominal), respectively.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.04 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.625 - 1.47 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.98 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 4.95 - 7.23 mg/L

Details on results:

- Exponential growth in the control: Yes, the biomass in the control cultures increased exponentially by a factor of > 16 within the 72 h test period corresponding to an average specific growth rate of 1.51 (required: > 0.92).
- Observation of abnormalities: No
- Effect concentrations exceeding solubility of substance in test medium: No

Reported statistics and error estimates:

The ErC50 and EyC50 values were calculated using ToxRat Standard Version 2.10 (ToxRat Solutions GmbH, Alsdorf, Germany). For the calculation of confidence intervals, values below 0% were set to 0.1% inhibition. Data were fit with the Logit regression model and the NOEC was determined by Dunnett´s test.

ANALYTICAL RESULTS

The LOQ of the method (0.5 mg/L) is higher than the two lowest test concentrations (0.100 and 0.316 mg/L). This has, however, no influence on the evaluation of the test.

HPLC measurements showed that the geometric mean measured test concentrations were within 91 - 105% of the nominal concentrations after 72 h, indicating that the test item was fully dissolved and stable for the duration of the test. Since test concentrations were maintained within ± 20% of the nominal concentrations throughout the test, effect concentrations were calculated based on nominal concentrations.

BIOLOGICAL RESULTS

The calculated median effect concentration ErC50 based on growth rate was 5.98 mg/L (95% CI: 4.95 - 7.23 mg/L), the ErC10 (72 h) was 1.04 mg/L (95% CI: 0.625 - 1.47 mg/L) and the NOErC (72 h) was 0.100 mg/L.

With respect to biomass production, the calculated EyC50 (72 h) was 1.28 mg/L (95% CI: 1.23 - 1.34 mg/L) and the NOEyC (72 h) was < 0.100 mg/L.

Table 1. Average specific growth rates between 0 and 72 h of exposure.

Nominal concentration [mg/L]	Replicate	GRa^a) µ3-0[d^-1]^b)	GRa^a) µ3-0[d^-1]^b) mean values	Coefficient of variation [%]	Inhibition [%]	Inhibition mean value [%]
Control	A	1.511	1.51 ^b)	0.1 ^c)	0.2 0.1 0.0 -0.1 0.0 -0.1	0.0
	B	1.513
	C	1.515
	D	1.517
	E	1.514
	F	1.517
0.100	A	1.505	1.50	0.5	0.6 1.5 0.6	0.9
	B	1.492
	C	1.506
0.316	A	1.477	1.48	0.0	2.5 2.5 2.6	2.5
	B	1.476
	C	1.476
1.00	A	1.341	1.34	0.3	11.5 11.0 11.3	11.3
	B	1.349
	C	1.343
3.16	A	0.981	0.99	1.2	35.2 33.7 35.0	34.6
	B	1.004
	C	0.984
10.0	A	0.686	0.66	4.7	54.7 58.8 56.7	56.7
	B	0.624
	C	0.656
31.6	A	-0.061	-0.06	0.0	104.0 104.0 104.0	104.0
	B	-0.061
	C	-0.061

^a)Average specific growth rate during the whole test period 0 – 3 d.

^b)The biomass in the control cultures increased exponentially by a factor of > 16 within the 72 h test period corresponding to an average specific growth rate of 1.51 (required: > 0.92).

^c)The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 0.1% (required: ≤ 7%).

Description of key information

ErC10 (72 h) = 1.04 mg/L (nominal, OECD 201, D. subspicatus)

ErC50 (72 h) = 5.98 mg/L (nominal, OECD 201, D. subspicatus)

Key value for chemical safety assessment

EC50 for freshwater algae:

5.98 mg/L

EC10 or NOEC for freshwater algae:

1.04 mg/L

Additional information

There is one key GLP study available investigating the toxic effects of the substance toward aquatic algae according to the OECD guideline 201.

In a static test, Desmodesmus subspicatus was exposed to the nominal test item concentrations of 0.1, 0.316, 1.0, 3.16, 10.0, and 31.6 mg/L under continuous light and controlled conditions for 72 h. A control was run in parallel. Since the test substance is soluble, the test solutions were prepared by the serial dilution of a 31.6 mg/L stock solution. The actual test concentrations were analytically verified by HPLC at the beginning (0 h), and after 24, 48 h and 72 h of exposure.

The geometric mean measured concentrations were 92 – 105% of the nominal concentration, showing that the test item was fully dissolved and stable throughout the duration of the test. Since the test concentrations were within ± 20% of the nominal concentrations, the effective concentrations (ErCx and EyCx) were calculated based on the nominal concentrations.

The calculated 72 h ErC10 was 1.04 mg/L (95% CI: 0.625 – 1.47 mg/L) and the ErC50 was 5.98 (95% CI: 4.95 – 7.23). The obtained NOErC (72 h) was 0.100 mg/L.