2'-methoxyacetoacetanilide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to current OECD-Guideline and GLP

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:

2'-methoxyacetoacetanilide

EC Number:

202-131-0

EC Name:

2'-methoxyacetoacetanilide

Cas Number:

92-15-9

Molecular formula:

C11H13NO3

IUPAC Name:

N-(2-methoxyphenyl)-3-oxobutanamide

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: One limit concentration of 100 mg/L
- Sampling method: The concentration of the test item was analytically determined from the limit concentration and the control after 0 h (test start) and 72 h (test end). Samples at start and end of the test were taken directly from the test replicates.

- Sample storage conditions before analysis:All samples were stored at 6 ± 2 °C until start of analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 200 mg/L, freshly prepared with dilution water, dispersion treatment was ultrasound for 30 minutes at 40 °C.
- Eluate: Dilution water
- Differential loading: one limit concentration of 100 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates
- Evidence of undissolved material (e.g. precipitate, surface film, etc): 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): 4 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

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22 °C, Min: 21 °C, Max: 23 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
100 7.99 8.07
Control 8.12 8.34

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Concentrations and Recovery Rates of Acetoacet-o-anisidid TTR

Sampling date 2009-06-09
0 h 2009-06-12
72 h
Start of analysis 2009-06-09 2009-06-12
Nominal concentration Acetoacet-o-anisidid TTR
Test item
[mg/L] Measured conc.
[mg/L] RR
[%] Measured conc.
[mg/L] RR
[%]
100 96.9 97 82.2 82
Control < LOQ — < LOQ —
Measured conc. = Measured concentration, mean value of 2 injections
RR = Recovery rate related to the nominal concentration Acetoacet-o-anisidid TTR
LOQ = Limit of quantification of the analytical method (0.09 mg/L)

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: 4878 cells/mL
- Control end cells density: Mean 576212 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] Specific Growth Rate Inhibition
[%] Yield Inhibition

[%]
100 -3 -20
10 0 1
1 1 6

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

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

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

EC-Values (0-72 h) of Acetoacet-o-anisidid TTR

                                based on the nominal test item concentration [mg/L]

                               

EC-Values	Yield inhibition
EyC10	> 100
EyC20	> 100
EyC50	> 100
	Rate-related inhibition
ErC10	> 100
ErC20	> 100
ErC50	> 100

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
100		1		4878		22693	133365	586497
		2		4878		20551	150334	591453
		3		4878		20631	142156	573296
		4		4878		22787	144366	554274
		5		4878		21554	141513	555590
		6		4878		23310	152850	537008
		Mean		4878		21921	144097	566353
Control		1		4878		25944	125394	610121
		2		4878		26413	125831	575644
		3		4878		26773	140032	566965
		4		4878		25304	146752	597542
		5		4878		22964	145859	583689
		6		4878		25737	141643	523308
		Mean		4878		25523	137585	576212

Evaluation after 72 h

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]			[%]
100	1		1.596	-0.40		581619	-1.80
	2		1.599	-0.57		586575	-2.67
	3		1.589	0.08		568418	0.51
	4		1.578	0.79		549396	3.84
	5		1.578	0.74		550712	3.61
	6		1.567	1.45		532130	6.86
	Mean		1.585	0.35		561475	1.73
Control	1		1.610			605243
	2		1.590			570766
	3		1.585			562087
	4		1.603			592664
	5		1.595			578811
	6		1.558			518430
	Mean		1.590			571334

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	1.671	1.576	1.582	1.610	0.053	3.32	9.87
	2	1.689	1.561	1.521	1.590	0.088	5.53
	3	1.703	1.654	1.398	1.585	0.164	10.3
	4	1.646	1.758	1.404	1.603	0.181	11.3
	5	1.549	1.849	1.387	1.595	0.234	14.7
	6	1.663	1.705	1.307	1.558	0.219	14.0
				Mean	1.590
				SD ±	0.02
				CV [%]	1.12

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the effects of the test item Acetoacet-o-anisidid TTR 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 100 mg/L. The EC50-values for specific growth rate inhibition (ErC50) and inhibition of yield (EyC50) after 72 h were > 100 mg/L.

Executive summary:

The toxicity of Acetoacet-o-anisidid TTR to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 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 nominally 2 - 5 x 10³ cells/mL. Six replicates were tested for the limit concentration of 100 mg/L and the control.

The concentrations of Acetoacet-o-anisidid TTR were analysed at all concentration levels after 0 and 72 h via HPLC analysis. The measured concentrations of Acetoacet-o-anisidid TTR at test start were in the range of 97 % of the nominal value. At the end of the test Acetoacet-o-anisidid TTR gave recoveries of 82 % of the nominal value. All effect values are given based on nominal concentrations of the test item.

Since the EC50-value for specific growth rate inhibition (ErC50) was > 100 mg/L there is no need for classification of the test item

EC-Values (0-72 h) of Acetoacet-o-anisidid TTR

                                based on the nominal test item concentration [mg/L]

                               

EC-Values	Yield inhibition
EyC10	> 100
EyC20	> 100
EyC50	> 100
	Rate-related inhibition
ErC10	> 100
ErC20	> 100
ErC50	> 100