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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2016 10 03 to 2016-10-07, with the definitive exposure phase from 2016-10-05 to 2016 10-07.

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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:

yes

Details on sampling:

Determination of the test item
No analytical monitoring was carried out, since no suitable method for the determination of the test item could be established.

Test solutions

Vehicle:

no

Details on test solutions:

Limit concentration
A saturated solution with a nominal concentration of 1.00 mg/L of the test item was tested.

Preparation of the saturated solution
The saturated solution (1.00 mg/L test item was weighed out) was prepared with dilution water (see Table 2) two days before the start of the exposure (at -48 hours). The test item was mixed with the dilution water by constant stirring with a magnetic stirrer at approximately 1100 rpm for 48 hours at room temperature. After completion of stirring, 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 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. The following filtrate, i.e. the saturated solution, was used as a limit concentration in the test. During filtration, the filter was always kept covered.

Control
Dilution water without test item incubated under the same conditions as the test group.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5)

Reason for the selectionof the test system
Daphnia magna is the preferred species in accordance with the
test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu),
14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.

Culture feeding
The culture daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

175 mg CaCO3/L

Test temperature:

19.9°C

pH:

Water Quality Parameters at the Start of the Exposure (0 hours)
(measured in one additional replicate of the saturated solution and the control)
Nominal
test item concentration
[mg/L] pH-value Dissolved O2 concentration [mg/L]
1.00* 7.82 3.22
Control 7.68 8.20
* = saturated solution


Water Quality Parameters at the End of the Exposure (48 hours)
(measured in all replicates of the saturated solution and the control)
Nominal
test item concentration
[mg/L] pH-values
Replicates
1 2 3 4
1.00* 7.57 7.51 7.50 7.55
Control 7.36 7.34 7.46 7.42
* = saturated solution

Dissolved oxygen:

Water Quality Parameters at the Start of the Exposure (0 hours)
(measured in one additional replicate of the saturated solution and the control)
Nominal
test item concentration
[mg/L] pH-value Dissolved O2 concentration [mg/L]
1.00* 7.82 3.22
Control 7.68 8.20
* = saturated solution

Water Quality Parameters at the End of the Exposure (48 hours)
(measured in all replicates of the saturated solution and the control)
Nominal
test item concentration
[mg/L] Dissolved O2 concentration [mg/L]]
Replicates
1 2 3 4
1.00* 7.60 7.57 7.09 7.10
Control 7.31 7.41 7.40 7.48
* = saturated solution

Conductivity:

492 µS/cm

Details on test conditions:

Test method
The study was performed with a static test design.

Test duration
48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume
20 mL in each test replicate (see ‘Application’)

Dilution water
Same as culture medium

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for the saturated solution and the control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization of the daphnids was not necessary, because the dilution water was equivalent to the culture medium.
 
Application
20 g test solution per replicate was weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by a pipette.

Test temperature (target)
18 - 22 °C, constant within ± 1 °C

Illumination (target)
Diffuse light, light intensity of max. 1500 lx

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

other: EC 10/50/100

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

other: EC 10/50/100

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The saturated solution was colorless and visually clear during the exposure period.

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2016 09-13 to 2016-09-14. For results of the most recent of the monthly performed reference tests.

EC50-Value (with 95% confidents limits) of the Reference Item Potassium dichromate based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 2.03 mg/L 0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000)
95% confidence limits 1.17 - 3.63 mg/L 0.6 - 2.1 mg/L, acc. to OECD 202 (2004)

Reported statistics and error estimates:

Methods of evaluation
The effects of the saturated solution were empirically derived from the observation data.
All effect levels (EC10 / 50 / 100) given were based on the nominal test item concentration, since analytical monitoring was not possible.

EC-values and statistical analyses for reference item
An EC50-value was calculated only for the reference item by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism5, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

The percentage of immobility, determined in the saturated solution and in the control after 24 and 48 hours under static conditions, is given in the Table.

 

Immobilization Ratesafter 24 and 48 hours ofExposure in the Definitive Test

 (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
1.00*	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

*    = saturated solution

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In the saturated solution with a nominal concentration of 1.00 mg/L of the test item 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol, no effects on Daphnia magna were observed.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of a saturated solution with a nominal concentration of 1.00 mg/L of the test item 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol (batch number: 3.April 2010) were determined at the test facility according to OECD 202 (2004) from 2016‑10‑03 to 2016-10-07, with the definitive exposure phase from 2016-10-05 to 2016‑10-07.

The limit test was conducted under static conditions over a period of 48 hours.The saturated solution was colorless and visually clear during the exposure period. Twenty daphnids, divided into 4 replicates with 5 daphnids each, were exposed to the saturated solution and the control.

No analytical monitoring was performed during the test period in agreement with the sponsor, since no suitable method for the determination of the poorly soluble test item could be established. Exposures are expressed in terms of the nominal concentration of the test item 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol.

The validity criteria of the test guideline were fulfilled.

 

 EC_{10 / 50 / 100}-Values

(based on the nominal concentration of the test item)

Effect values	Test duration [h]	1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol Nominal test item concentration [mg/L]
EC10 / 50 / 100	24	> 1.00
	48	> 1.00