N-(2-methoxy-5-methylphenyl)acetamide

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:

2009-08-10 to 2009-08-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with 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:

N-(2-methoxy-5-methylphenyl)acetamide

EC Number:

230-157-2

EC Name:

N-(2-methoxy-5-methylphenyl)acetamide

Cas Number:

6962-44-3

Molecular formula:

C10H13NO2

IUPAC Name:

N-(2-methoxy-5-methylphenyl)acetamide

Details on test material:

- Name of test material (as cited in study report): Acetkresidin TTRS
- Molecular formula: C10H13NO2
- Molecular weight: 179.22 g/mol
- Physical state: Solid, grey flakes
- Analytical purity: 98.60 % (a/a) comp.1
- Purity test date: 2008-07-09
- 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 via HPLC-DAD analysis.
- Sampling method: Samples at test start and end 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: A stock solution of 200 mg/L was freshly prepared with dilution water
- Eluate: Dilution water
- Differential loading: Limit test concentration of 100 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

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:

None

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 7.93 8.59
Control 7.92 8.99

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information on materials and methods

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 1220216 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 1 8
1 4 19

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

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:

The NOEC and LOEC were determined by calculation of statistical significance of growth rates and yield.
t-test was carried out for the determination of statistically significant differences compared to control replicates. When running a t-test a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The a-value was a=0.05.
EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).

Any other information on results incl. tables

NOEC, LOEC andEC-Values of Acetkresidin TTRS (0-72 h)

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

                               

EC-Values	Rate-related inhibition
NOEC	100
LOEC	> 100
ErC10	> 100
ErC20	> 100
ErC50	> 100
	Yield inhibition
NOEC	100
LOEC	> 100
EyC10	> 100
EyC20	> 100
EyC50	> 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		5074		37538	214728	1090049
		2		5074		42704	248671	1281224
		3		5074		36824	217794	1053899
		4		5074		46484	244186	1213959
		5		5074		46566	260213	1333232
		6		5074		45549	239606	1218555
		Mean		5074		42611	237533	1198486
Control		1		5074		31531	229356	1127609
		2		5074		34918	208545	1178556
		3		5074		32064	228483	1114706
		4		5074		44541	262074	1413715
		5		5074		40588	230504	1271006
		6		5074		35525	226712	1215702
		Mean		5074		36528	230946	1220216

Evaluation after 72 h

                        Statistically significant differences of growth rates and yield compared to

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

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		1.790	2.00		1084975	10.71
	2		1.844	-0.95		1276150	-5.02
	3		1.779	2.61		1048825	13.69
	4		1.826	0.03		1208885	0.51
	5		1.857	-1.68		1328158	-9.30
	6		1.827	-0.04		1213481	0.14
	Mean	(-)	1.820	0.33	(-)	1193412	1.79
Control	1		1.801			1122535
	2		1.816			1173482
	3		1.797			1109632
	4		1.877			1408641
	5		1.841			1265932
	6		1.826			1210628
	Mean		1.826			1215142

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	1.827	1.984	1.593	1.801	0.1971	10.9	9.97
	2	1.929	1.787	1.732	1.816	0.1016	5.60
	3	1.844	1.964	1.585	1.797	0.1936	10.8
	4	2.172	1.772	1.685	1.877	0.2597	13.8
	5	2.079	1.737	1.707	1.841	0.2068	11.2
	6	1.946	1.853	1.679	1.826	0.1354	7.41
				Mean	1.826
				SD ±	0.03
				CV [%]	1.61

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the effects of the test item Acetkresidin TTRS 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. The NOEC for both the specific growth rate inhibition and the yield inhibition was 100 mg/L, the LOEC was > 100 mg/L. All effect values are given based on nominal concentrations of the test item.

Executive summary:

The toxicity of Acetkresidin TTRS to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201. 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 nominally 2 - 5 x 10³ cells/mL. Six replicates were tested for the limit concentration of 100 mg/L and the control. Environmental conditions were determined to be within the acceptable limits.
The concentration of Acetkresidin TTRS was determined at test start and end via HPLC-DAD analysis. The measured concentration at test start and end was 95 % and 92 % of the nominal value, respectively. For details of the analytical results please refer to section 4.2.3. All effect levels are given based on the nominal concentration of the test item.

Table 1:   NOEC, LOEC and EC-Valuesof Acetkresidin TTRS(0-72 h)

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

                               

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