Propanamide, N-[4-(2,4-dihydroxyphenyl)-2-thiazolyl]-2-methyl-

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:

18 Feb - 21 Feb 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Test material

Test material information

Constituent 1

Reference substance name:

N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide

EC Number:

807-621-3

Cas Number:

1428450-95-6

Molecular formula:

C13H14N2O3S

IUPAC Name:

N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 0, 0.46, 1.0, 2.2, 4.6 and 10 mg/L

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution containing 10.5 mg/L in nutrient medium (demineralised water enriched with minerals but without algae) was prepared. For this the nominal load of test item was solved with the help of an ultrasonic bath. Because after 30 min the resulting solution was not complete clear, the solution was filtrated through 0.45 µm Teflon filters. The lower treatments were prepared by dilution of the stock solution with nutrient medium.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 86.81 (SAG Strain Number)
- Source: The culture of Desmodesmus subspicatus was obtained in January 2013 from MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen, Germany). The algae are kept as stock culture on solid agar at 7 °C.

ACCLIMATION
- Acclimation period: Four days before the start of the test, an aliquot of the permanent culture was brought into pre-culture medium and incubated for 96 h. The resulting culture is growing exponentially.
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23 °C

pH:

7.7 - 8.2

Nominal and measured concentrations:

nominal: control, 0.46, 1.0, 2.2, 4.6 and 10 mg/L
measured: 0, 0.39, 0.72, 1.49, 1.87, 6.91 mg/L (based on the geometric mean measured concentration)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Material, size, headspace, fill volume: glass, 65 mL, headspace: 20 ± 1 mL, fill volume: 45 ± 1 mL
- Aeration: shaken on an orbital shaker
- Initial cells density: 3822 cells/mL
- Control end cells density: 227197 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: demineralised water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: constant illumination
- Light intensity and quality: 5800 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Before the start and every 24 h, the cell number was calculated based on the determination of the light absorption at 440 nm. At the end of the test, the treatments were examined microscopically in order to assess the appearance of the alga and detect abnormalities.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.39 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.39 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.39 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.68 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.73 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): small cells were observed at a nominal concentration of 2.2 mg/L
- Any stimulation of growth found in any treatment: yes, No inhibition but instead stimulation was observed at the nominal concentration of 0.46 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: EC50 (72 h) 0.89 mg/L based on growth rate; in the range of interlaboratory test (0.60 - 1.03 mg/L)

Reported statistics and error estimates:

NOEC/LOEC determination: t-test
ECx determination: Software OriginTM. The values for p resp. χ² were calculated. The data were evaluated using sigmoidal fit using a logistic equation.

Any other information on results incl. tables

Table 1: Cell numbers

Nominal concentration [mg/L]	Measured concentration [mg/L] (geometric mean)	Replicate	Cell number/mL
			0 h	24 h	48 h	72 h
Control	Control	1	3822	14014	49686	198744
		2	3822	14014	53508	212758
		3	3822	12740	54782	256074
		4	3822	15288	54782	235690
		5	3822	14014	50960	219128
		6	3822	12740	54782	240786
		Mean	3822	13802	53083	227197
		SD	0	959	2231	20851
0.46	0.39	1	3822	14014	64974	321048
		2	3822	11466	64974	308308
		3	3822	14014	53508	284102
		Mean	3822	13165	61152	304486
		SD	0	1471	6620	18767
1.0	0.72	1	3822	14014	34398	133770
		2	3822	14014	38220	146510
		3	3822	15288	31850	122304
		Mean	3822	14439	34823	134195
		SD	0	736		12109
2.2	1.49	1	3822	8918	16562	30576
		2	3822	10192	17836	35672
		3	3822	10192	19110	44590
		Mean	3822	9767	17836	36946
		SD	0	736	1274	7093
4.6	1.87	1	3822	11466	14014	33124
		2	3822	7644	15288	29302
		3	3822	8918	16562	29302
		Mean	3822	9343	15288	30576
		SD	0	1946	1274	2207
10	6.91	1	3822	6370	14014	24206
		2	3822	6370	12740	19110
		3	3822	6370	16562	28028
		Mean	3822	6370	14439	23781
		SD	0	0	1946	4474

 

 

 

 

 

Table 2: Inhibition of growth

Nominal concentration [mg/L]	Measured concentration [mg/L] (geometric mean)	Replicate	Inhibition Growth Rate µ
			72 h
Control	Control	1	3.19
		2	1.522
		3	-3.02
		4	-0.99
		5	0.80
		6	-1.51
		Mean	0!
		SD	2.26
0.46	0.39	1	-8.56
		2	-7.57
		3	-5.56
		Mean	-7.23
		SD	1.53
1.0	0.72	1	12.89
		2	10.66
		3	15.09
		Mean	12.88
		SD	2.21
2.2	1.49	1	49.05
		2	45.25
		3	39.81
		Mean	44.71
		SD	4.65
4.6	1.87	1	47.09
		2	50.09
		3	50.09
		Mean	49.09
		SD	1.73
10	6.91	1	54.78
		2	60.57
		3	51.18
		Mean	55.51
		SD	4.73

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A NOEC of 0.39 mg/L and a LOEC of 0.72 mg/L was statistically determined for the endpoints.
The EC10s were determined as 0.33 mg/L (Growth Rate), 0.51 mg/L (AUC) and
0.68 mg/L (Yield). The EC50s were determined as 3.3 mg/L (Growth Rate), 1.0 mg/L
(AUC) and 0.73 mg/L (Yield).

Executive summary:

One experiment was performed.

The study was performed using five concentrations ranging from 0.46 to 10 mg/L nominal

concentration. Incubation time (test system Desmodesmus subspicatus) was 72 hours.

The cell concentration of each replicate was determined by measuring the absorption of

the solutions at 440 nm every 24 hours with a spectral photometer. The cell density of the

cultures was calculated based on the correlation curve between the adsorption and the cell

density of the cultures determined by microscope counts. Growth rate μ, area under the

growth curve (AUC1) and the yield2 were determined from the cell densities at the respective

observation times.

Significant inhibition of algal growth was observed at the following concentrations:

1 / 2.2 / 4.6 / 10 mg/L

At the start and at the end of the test, the content of the test item in the test solutions was

determined using HPLC-UV. The measured concentrations lay in the range of 37 % and

99 % of the nominal concentrations. Therefore, the determination of the results was based

on the geometric mean of the measured concentrations.

The EC50s of potassium dichromate were tested in a separate reference test. The values

lay within the normal range of the laboratory.