N-(2-ethoxyphenyl)-N'-(4-isododecylphenyl)oxamide

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:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD guideloine and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The limit test item concentration and control were analytically verified via UPLC-MS/MS after 0 hours (test start) and 72 hours (test end).
- Sampling method: Samples at test start were taken from additional replicates without algae. After 72 hours samples were taken directly from pooled test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared with dilution water.
- Eluate: DIlution water
- Differential loading: None, saturated solution corresponding to a geometric mean measured test item concentration of 36.4 µg/L.
- Controls: Six replicates withou test item were tested under the same test conditions as the test replicates.

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 every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 21.0 max.: 22.5, mean value: 21.8

pH:

Geometric mean measured test item concentration pH-value
[µg/L] Start; 0 hours End; 72 hours
36.4 7.47 7.84
Control 7.45 7.95

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- 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: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 4876 cells/mL
- Control end cells density: 819255 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6



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, mean value: 69.2



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:
Results of the Range Finding Test (0-72 hours)

Saturated solution
[%] Specific Growth Rate Inhibition[%] Yield Inhibition[%]
100 -3 -16
10 1 6
1 5 21

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

36.4 µg/L

Nominal / measured:

meas. (geom. mean)

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
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.488 0.699 ± 0.471
95 % confidence interval 0.466 – 0.512
Yield inhibition
EyC50 0.291 0.318 ± 0.147
95 % confidence interval 0.274 – 0.328
SD = Standard deviation

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. t-test was used for NOEC/LOEC calculation. When running a t-test a Normality test and an Equal Variance test was done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05. Normality test failed for both calculations of growth rate and yield. No transformation was found to pass Normality test.

NOEC, LOEC, EC - values of Test item (0-72 hours)

                   based on geometric mean measured test item concentrations [µg/L]

	Inhibition of Growth Rate
NOEC	36.4
LOEC	> 36.4
ErC10	> 36.4
ErC20	> 36.4
ErC50	> 36.4
	Inhibition of Yield
NOEC	36.4
LOEC	> 36.4
EyC10	> 36.4
EyC20	> 36.4
EyC50	> 36.4

Cell Densities

	Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[µg/L]		No.		0 hours		24 hours	48 hours	72 hours
36.4		1		4876		28364	178584	1022197
		2		4876		28344	177690	808129
		3		4876		29485	192225	876946
		4		4876		31749	186667	880298
		5		4876		24627	174390	879519
		6		4876		28259	170947	912150
		Mean		4876		28471	180084	896540
Control		1		4876		21266	94751	528562
		2		4876		23717	156226	857043
		3		4876		28107	159320	902943
		4		4876		26320	173855	849494
		5		4876		27091	167701	879790
		6		4876		28263	175155	897696
		Mean		4876		25794	154501	819255

Evaluation after 72 hours

                   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		Growth rate inhibition	Yield		Inhibition of yield
[µg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
36.4	1		1.78	-4.65		1017321	-24.9
	2		1.70	-0.05		803253	1.37
	3		1.73	-1.65		872070	-7.08
	4		1.73	-1.72		875422	-7.50
	5		1.73	-1.71		874643	-7.40
	6		1.74	-2.42		907274	-11.4
	Mean	(-)	1.74	-2.03	(-)	891664	-9.49
Control	1		1.56			523686
	2		1.72			852167
	3		1.74			898067
	4		1.72			844618
	5		1.73			874914
	6		1.74			892820
	Mean		1.70			814379

Section-by-Section and Average Specific Growth Rates of the Control Group
(0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.47	1.49	1.72	1.56	0.136	8.73	6.25
	2	1.58	1.89	1.70	1.72	0.153	8.86
	3	1.75	1.74	1.74	1.74	0.0097	0.560
	4	1.69	1.89	1.59	1.72	0.154	8.93
	5	1.72	1.82	1.66	1.73	0.0840	4.85
	6	1.76	1.82	1.63	1.74	0.0964	5.54
				Mean	1.70
				SD ±	0.07
				CV [%]	4.08

Validity criteria fulfilled:

yes

Conclusions:

In this study no inhibition on the growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item. The NOEC for inhibition of specific growth rate and yield after 72 hours was 36.4 µg/L.
All effect levels are given based on the geometric mean measured test item concentration of Hostavin 3206 (impoverished of xylene) which has been analytically verified by UPLC-MS/MS.

Executive summary:

The toxicity of the test item 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 over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4876 cells/mL. Based on a preliminary range finding the saturated solution* was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

The saturated solution* and the control were analysed by UPLC-MS/MS after (fresh media) and 72 h (old media).

The initially measured concentration (fresh media, 0 h) of the test item in the freshly prepared saturated solution* was 40.9 µg/L, representing the maximum solubility of the test item under test conditions. The recovery rate of the test item in the old media (72 h) was 79 % of the initially measured concentration, demonstrating that the concentration of the test item had decreased for the duration of the test. All effect values given are based on the geometric mean measured test item concentration

NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on geometric mean measured test item concentrations [µg/L]

	Inhibition of Growth Rate
NOEC	36.4
LOEC	> 36.4
ErC10	> 36.4
ErC20	> 36.4
ErC50	> 36.4
	Inhibition of Yield
NOEC	36.4
LOEC	> 36.4
EyC10	> 36.4
EyC20	> 36.4
EyC50	> 36.4

Description of key information

In this study no inhibition on the growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item. The NOEC for inhibition of specific growth rate and yield after 72 hours was 36.4 µg/L. (saturated solution)