Ethylene diacetoacetate

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:

2009-06-13 to 2009-06-16

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene diacetoacetate

EC Number:

226-724-9

EC Name:

Ethylene diacetoacetate

Cas Number:

5459-04-1

Molecular formula:

C10H14O6

IUPAC Name:

ethane-1,2-diyl bis(3-oxobutanoate)

Details on test material:

- Name of test material (as cited in study report): Acetessigsäureglycolester
- Molecular formula: C10H14O6
- Molecular weight: 230.22 g/mol
- Physical state: Liquid
- Analytical purity: 99.22 % (w/w)
- Storage condition of test material: Room temperature, protected from moisture and light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The limit concentration and the control were analytically verified at the beginning and the end of the test.
- Sampling method: Separate replicates for the test item analysis after 0 h (all concentrations without algae) were prepared. At the end of the test samples were taken directly from the test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 +/- 2 °C if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 200 mg/L was freshly prepared with dilution water and agitated
- Eluate: Dilution water
- Differential loading: Limit test concentration 100 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22.5 °C, Min: 22 °C, Max: 23 °C

pH:

Geometric mean measured
test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
76.0 7.85 8.21
Control 7.91 8.52

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Concentrations and Recovery Rates of Acetessigsäureglycolester

Sampling date 2009-07-13
Test start 2009-07-16
Test end
Start of analysis 2009-07-13
2009-07-16

Nominal concentration of the Acetessigsäureglycolester
Geometric mean measured concentration
test item

[mg/L] Meas.
conc.
[mg/L] RR

[%] Meas.
conc.
[mg/L] RR

[%] Test item

[mg/L]
100 119 119 48.5 49 76.0
Control < LOQ — < LOQ — < LOQ

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 5074 cells/mL
- Control end cells density: Mean 1127529 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0-72 h)
Nominal Test Item Concentration
[mg/L] Rate-related inhibition
[%] Yield Inhibition

[%]
100 0 1
10 0 -2
1 1 5

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

76 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: Yes

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.695 (CI 0.657 - 0.729) mg/L
Yield Inhibition: 0.320 (CI 0.294 - 0.358) mg/L

Reported statistics and error estimates:

All calculations were based on the fluorescence values of each replicate.

Any other information on results incl. tables

NOEC, LOEC and EC-Values of Acetessigsäureglycolester(0-72 h)

                                based on geometric mean measured concentration of the test item [mg/L]

                               

EC-Values	Rate-related inhibition
NOEC	76.0
LOEC	> 76.0
ErC10	> 76.0
ErC20	> 76.0
ErC50	> 76.0
	Yield inhibition
NOEC	76.0
LOEC	> 76.0
EyC10	> 76.0
EyC20	> 76.0
EyC50	> 76.0

Cell Densities

	Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
76.0		1		5074		58464	326047	1386389
		2		5074		56029	337863	1423518
		3		5074		49223	326141	1351100
		4		5074		51855	338453	1367361
		5		5074		47788	361749	1348226
		6		5074		55967	370040	1369218
		Mean		5074		53221	343382	1374302
Control		1		5074		38940	275945	1287780
		2		5074		39047	238032	1029201
		3		5074		42072	284583	1092997
		4		5074		37694	265486	1100077
		5		5074		43905	261468	1085481
		6		5074		43286	285563	1169638
		Mean		5074		40824	268513	1127529

Evaluation after 72 h

                        Statistically significant differences of growth rates and yield compared to

                                control values are marked (+), not significant differences are marked (-).

Geometric mean measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
76.0	1		1.870	-3.87		1381315	-23.06
	2		1.879	-4.36		1418444	-26.37
	3		1.862	-3.40		1346026	-19.92
	4		1.866	-3.62		1362287	-21.37
	5		1.861	-3.36		1343152	-19.66
	6		1.866	-3.64		1364144	-21.53
	Mean	(+)*	1.867	-3.71	(+)*	1369228	-21.99
Control	1		1.846			1282706
	2		1.771			1024127
	3		1.791			1087923
	4		1.793			1095003
	5		1.789			1080407
	6		1.813			1164564
	Mean		1.800			1122455

Section-by-Section and Average Specific Growth Rates of the Control Group

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 – 24 h	24 – 48 h	48 – 72 h
Control	1	2.038	1.958	1.540	1.846	0.2672	14.5	18.7
	2	2.041	1.808	1.464	1.771	0.2900	16.4
	3	2.115	1.912	1.346	1.791	0.3988	22.3
	4	2.005	1.952	1.422	1.793	0.3228	18.0
	5	2.158	1.784	1.423	1.789	0.3672	20.5
	6	2.144	1.887	1.410	1.813	0.3723	20.5
				Mean	1.800
				SD ±	0.03
				CV [%]	1.44

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the effects of the test item Acetessigsäureglycolester on the growth of the freshwater green alga Desmodesmus subspicatus were tested. No inhibiting effects on specific growth rate and yield were found in the limit concentration of nominal 100 mg/L, corresponding to geoemtric mean measured concentration of 76.0 mg/L. The EC50-values for specific growth rate inhibition (ErC50) and inhibition of yield (EyC50) after 72 h were > 76.0 mg/L. The NOEC for both parameters was 76 mg/L, the LOEC was > 76 mg/L.

Executive summary:

The toxicity of Acetessigsäureglycolester(Batch no.: DEB2 058115) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorien in 31157 , from July 13 to 16, 2009. The aim of the study was to assess the effects on growth rate and yield production over a period of 72 h in a limit test.
The study was conducted under static conditions with an initial cell density of approximately 2 - 5 x 10³ cells/mL. One limit concentration of 100 mg/L was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.
The concentration of Acetessigsäureglycolester was analysed at test start and end via LC-MS/MS analysis. Measured concentrations at test start met the nominal ranges. At the end of the test the limit concentration gave a recovery of 49 % of the nominal value. All effect levels are given based on geometric mean measured concentration of the test item

NOEC, LOEC and EC-Values of Acetessigsäureglycolester(0-72 h)

                                based on geometric mean measured concentration of the test item [mg/L]

                               

EC-Values	Rate-related inhibition
NOEC	76.0
LOEC	> 76.0
ErC10	> 76.0
ErC20	> 76.0
ErC50	> 76.0
	Yield inhibition
NOEC	76.0
LOEC	> 76.0
EyC10	> 76.0
EyC20	> 76.0
EyC50	> 76.0