2,2'-(1,4-phenylene)bis[4-[(4-methoxyphenyl)methylene]oxazol-5(4H)-one]

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:

2016-09-10 to 2016-09-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(1,4-phenylene)bis[4-[(4-methoxyphenyl)methylene]oxazol-5(4H)-one]

EC Number:

257-055-0

EC Name:

2,2'-(1,4-phenylene)bis[4-[(4-methoxyphenyl)methylene]oxazol-5(4H)-one]

Cas Number:

51202-86-9

Molecular formula:

C28H20N2O6

IUPAC Name:

2,2'-(1,4-phenylene)bis[4-(4-methoxybenzylidene)-1,3-oxazol-5(4H)-one]

Test material form:

solid: particulate/powder

Details on test material:

Expiry date
2026-04-19 (Statement of producer)

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
The saturated solution (1.00 mg/L test item was weighed out) was prepared with demineralized water two days prior to the start of the exposure (0 h). The dispersion was stirred for 48 hours (1100 rpm, room temperature) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded.
The filtration was interrupted for ca. 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. After the separation treatment, the components of the dilution water were added to the saturated solution. The following filtrate, i.e. the saturated solution, was used in the test. During filtration the filter was always kept covered.

Test concentration
The saturated solution was tested in a limit test.

Control
Six replicates (without test item) were exposed under the same conditions as the test item replicates.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 21.5 max.: 23.0 mean value: 22.2

pH:

Nominal Test Item Loading pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.84 8.91
Control 8.02 9.12

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

No analytical monitoring

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 10^3 cells/mL, Actual: mean 6305 cells/mL
- Control end cells density: Mean 1661567 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:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5707 lux,



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.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

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

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2016-04-26 to 2016-04-29 (with headspace). The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.460 0.780 ± 0.473
95% confidence interval 0.443 – 0.481
Yield inhibition
EyC50 0.317 0.411 ± 0.276
95% confidence interval 0.208 – 0.347
SD = Standard deviation

Reported statistics and error estimates:

EL-values and statistical analyses
EL-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).

NOEL and LOEL values
NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. As a standard, a t-test was used for NOEL/LOEL calculations. When running a t-test a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normal distribution of data. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.


Software
The data for the tables in this report are computer generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.

Any other information on results incl. tables

Cell Densities

Nominal Test Item Loading	Replicate	Cell Density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
1.00	1	6305	27806	198248	1655369
	2	6305	27191	191888	1606713
	3	6305	35800	230599	1689536
	4	6305	26681	206773	1652663
	5	6305	29426	205589	1646348
	6	6305	30577	211768	1501959
	Mean	6305	29580	207478	1625431
Control	1	6305	30176	226576	1715617
	2	6305	31425	218925	1721368
	3	6305	33139	235963	1848730
	4	6305	27501	211150	1610581
	5	6305	28741	200545	1540219
	6	6305	30343	235958	1532889
	Mean	6305	30221	221520	1661567

 

 

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 

Nominal Test Item Loading		Replicate		Growth Rate			Inhibition of Growth Rate		Yield			Inhibition of Yield
[mg/L]		No.		[d^-1]			[%]		[cells/mL]			[%]
1.00		1				1.86		0				1649064		0
		2				1.85		1				1600408		3
		3				1.86		0				1683231		-2
		4				1.86		0				1646358		1
		5				1.86		0				1640043		1
		6				1.82		2				1495654		10
		Mean		(-)		1.85		0		(-)		1619126		2
Control		1				1.87						1709312
		2				1.87						1715063
		3				1.89						1842425
		4				1.85						1604276
		5				1.83						1533914
		6				1.83						1526584
		Mean				1.86						1655262

 

negative inhibition = increase of growth

 

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

	Replicate No.	Specific Growth Rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.57	2.02	2.02	1.87	0.263	14.0	13.9
	2	1.61	1.94	2.06	1.87	0.236	12.6
	3	1.66	1.96	2.06	1.89	0.208	11.0
	4	1.47	2.04	2.03	1.85	0.325	17.6
	5	1.52	1.94	2.04	1.83	0.278	15.1
	6	1.57	2.05	1.87	1.83	0.242	13.2
				Mean	1.86
				SD ±	0.02
				CV [%]	1.32

SD = Standard deviation  CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Polysynthren-Gelb GG was found not to inhibit the growth rate of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours. The NOEL-values of the test item for both inhibition of growth rate and inhibition of yield after 72 hours were 1.00 mg/L. The EL50-values of the test item for both inhibition of the growth rate (ErL50) and inhibition of yield (EyL50) after 72 hours were > 1.00 mg/L.
All effect values are given based on the nominal loading of Polysynthren-Gelb GG.

Executive summary:

The toxicity of Polysynthren-GelbGG (Batch no.:DEB2259062 ag) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 Method C.3 from2016-09-10 to 2016-09-15, with the definitive exposure phase from 2016-09-12 to 2016-09-15at the test facility.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 6305 cells/mL. A saturated solution with a nominal loading of 1.00 mg/L was freshly prepared with demineralized water two days prior to the start of exposure. The dispersion was stirred for 48 hours at room temperature (1100 rpm). After stirring, undissolved particles were removed by membrane filtration. After the separation treatment, the components of the dilution water were added to the saturated solution. The saturated solution was tested in a limit test. Six replicates were tested for the limit concentration and the control. The environmental conditions were within the acceptable limits.

 

The test media were visually clear throughout the test period. No analytical determination was carried out since no suitable method for the determination of the test item could be established. Therefore, all effect values given are based on the nominal loading ofPolysynthren-Gelb GG.

 

 NOEL, LOEL, EL-values of Polysynthren-GelbGG(0 - 72 hours)

                  based on the nominal loading of the test item [mg/L]

	Inhibition of Growth Rate nominal test item loading [mg/L]
NOEL	1.00
LOEL	> 1.00
ErL10	> 1.00
ErL20	> 1.00
ErL50	> 1.00
	Inhibition of Yield nominal test item loading [mg/L]
NOEL	1.00
LOEL	> 1.00
EyL10	> 1.00
EyL20	> 1.00
EyL50	> 1.00