1-[(4-methoxy-2-nitrophenyl)azo]-2-naphthol

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:

2017-11-17 to 2017-12-07

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

1-[(4-methoxy-2-nitrophenyl)azo]-2-naphthol

EC Number:

256-458-9

EC Name:

1-[(4-methoxy-2-nitrophenyl)azo]-2-naphthol

Cas Number:

49744-28-7

Molecular formula:

C17H13N3O4

IUPAC Name:

1-[(4-methoxy-2-nitrophenyl)diazenyl]-2-naphthol

Test material form:

solid: particulate/powder

Details on test material:

Batch number
3. April 2010

CAS number
49744-28-7

Sum formula
C17H13N3O4

Molecular weight
323.31 g/mol

Purity of main component (certified)
98.50% (w/w) (comp. 1)


Water solubility
3.3 µg/L

Appearance
Red powder

pH value
9.1 (10% H2O)

Stability
Stable in water and light at room temperature

Expiry date
2020-04-03

Recommended storage
Room temperature

Sampling and analysis

Analytical monitoring:

no

Remarks:

No analytical monitoring was carried out due to the low solubility of the test item .

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
The saturated solution (1 mg/L test item was weighed out) was prepared with demineralized water three days prior to the start of the exposure (0 h). The dispersion was stirred for 72 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 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 concentrations (nominal)
Five dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2.50: 2.56 - 6.40 - 16.0 - 40.0 - 100% of the saturated solution. The dilution levels are based on the results of a preliminary range finding test (non-GLP, Erlenmeyer flasks, with headspace in OECD TG 201 medium)

Control
Six replicates (without test item) were tested under the same test 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.: 22.0 max.: 23.0 mean value: 22.5

pH:

Nominal test item concentration pH-value
[% saturated solution] Start; 0 hours End; 72 hours
100 7.74 9.09
40.0 7.73 9.32
16.0 7.80 9.25
6.40 7.86 9.16
2.56 8.06 9.19
Control 8.17 9.16

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

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 6839 cells/mL
- Control end cells density: Mean 2253449 cells/mL
- 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:
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 0.064a2EDTA 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: 6132 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test at the saturated solution.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate und yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

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: The test media clear throughout the exposure period.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA-ALDRICH, 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 2017-10-10 to 2017-10-13. 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 0.535 0.745 ± 0.524
95 % confidence interval 0.523 – 0.547
Yield inhibition
EyC50 0.320 0.398 ± 0.291
95 % confidence interval 0.249 – 0.339
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statisticall analyses
EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were determined from the recorded data without any statistical calculations.

NOEC, LOEC and statistically analyses
The NOEC and LOEC were determined by calculation of significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) was used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. 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. The Normality test failed for calculation of growth rate. Therefore, the data were transformed (Y=10Y).

Software
The data for the tables in this report were 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.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- ToxRat Professional, Version 3.2, TOXRAT SOLUTIONS GMBH

Any other information on results incl. tables

Cell Densities

Nominal test item concentration   [% saturated solution]		Replicate		Cell density [cells/mL]
		No.		0 hours		24 hours		48 hours		72 hours
100		1		6839		28927		339712		2287761
		2		6839		28389		297449		2252298
		3		6839		34182		329694		2450740
		Mean		6839		30499		322285		2330266
40.0		1		6839		33293		353078		2529683
		2		6839		35394		402668		2771546
		3		6839		32834		390145		2609095
		Mean		6839		33840		381964		2636775
16.0		1		6839		35160		421687		2508733
		2		6839		32918		389911		2402812
		3		6839		33217		435369		2696933
		Mean		6839		33765		415656		2536159
6.40		1		6839		28117		344827		1819367
		2		6839		31455		359914		2545015
		3		6839		29185		348385		2197933
		Mean		6839		29586		351042		2187438
2.56		1		6839		34235		355782		2271083
		2		6839		29750		344488		2284660
		3		6839		27751		341304		2256336
		Mean		6839		30579		347191		2270693
Control		1		6839		28881		252752		1931550
		2		6839		39935		459901		2485266
		3		6839		33302		373923		1288708
		4		6839		31657		388705		1934710
		5		6839		33226		457349		2978824
		6		6839		33846		385276		2901636
		Mean		6839		33475		386318		2253449

 

 

 

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 concentration		Replicate		Growth rate			Inhibition of growth rate		Yield			Inhibition of yield
[% saturated solution]		No.		[d^-1]			[%]		[cells/mL]			[%]
100		1				1.94		-1				2280922		-2
		2				1.93		-1				2245459		0
		3				1.96		-2				2443901		-9
		Mean		(-)		1.94		-1		(-)		2323427		-3
40.0		1				1.97		-3				2522844		-12
		2				2.00		-4				2764707		-23
		3				1.98		-3				2602256		-16
		Mean		(-)		1.99		-3		(-)		2629936		-17
16.0		1				1.97		-3				2501894		-11
		2				1.95		-2				2395973		-7
		3				1.99		-4				2690094		-20
		Mean		(-)		1.97		-3		(-)		2529320		-13
6.40		1				1.86		3				1812528		19
		2				1.97		-3				2538176		-13
		3				1.92		0				2191094		2
		Mean		(-)		1.92		0		(-)		2180599		3
2.56		1				1.94		-1				2264244		-1
		2				1.94		-1				2277821		-1
		3				1.93		-1				2249497		0
		Mean		(-)		1.94		-1		(-)		2263854		-1
Control		1				1.88						1924711
		2				1.97						2478427
		3				1.75						1281869
		4				1.88						1927871
		5				2.03						2971985
		6				2.02						2894797
		Mean				1.92						2246610

 

 

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	1.44	2.17	2.03	1.88	0.388	20.6	25.4
	2	1.77	2.44	1.69	1.97	0.416	21.2
	3	1.58	2.42	1.24	1.75	0.607	34.8
	4	1.53	2.51	1.61	1.88	0.544	28.9
	5	1.58	2.62	1.87	2.03	0.537	26.5
	6	1.60	2.43	2.02	2.02	0.417	20.7
				Mean	1.92
				SD ±	0.11
				CV [%]	5.50

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 % saturated solution, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 100 % saturated solution, respectively. The saturated solution was prepared with a nominal loading of 1 mg/L.

Executive summary:

The toxicity of 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol (batch number:3.April 2010 g) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2017-11-17 to 2017-12-07 at 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 6839 cells/mL. A saturated solution with a nominal loading of 1.00 mg test item/L was tested. Five concentrations were tested in a geometrical series with a dilution factor of 2.50 (nominal):2.56 - 6.40 - 16.0 - 40.0 - 100 % saturated solution. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

 

No analytical monitoring was carried out due to the low solubility of the test item.

 

All effect values were based on the nominal test item of1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol. The results are summarized in the following table:

 

NOEC, LOEC, EC - values of 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol (0-72hours)

                  based on the nominal test item concentration [% saturated solution]

 

	Nominal test item concentration [% saturated solution]
	Inhibition of Growth Rate
NOEC	100
LOEC	> 100
ErC10	> 100
ErC20	> 100
ErC50	> 100
	Inhibition of Yield
NOEC	100
LOEC	> 100
EyC10	> 100
EyC20	> 100
EyC50	> 100