Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, 4-[(5-chloro-2-hydroxyphenyl)azo]-4,5-dihydro-3-methyl-1-phenyl-3H-pyrazol-3-one 4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-1-phenyl-3H-pyrazol-3-one 3-[[1-[[(2-ethylhexyl)amino]carbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzoate cobaltate complexes

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, 4-[(5-chloro-2-hydroxyphenyl)azo]-4,5-dihydro-3-methyl-1-phenyl-3H-pyrazol-3-one 4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-1-phenyl-3H-pyrazol-3-one 3-[[1-[[(2-ethylhexyl)amino]carbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzoate cobaltate complexes

EC Number:

276-160-2

EC Name:

Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, 4-[(5-chloro-2-hydroxyphenyl)azo]-4,5-dihydro-3-methyl-1-phenyl-3H-pyrazol-3-one 4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-1-phenyl-3H-pyrazol-3-one 3-[[1-[[(2-ethylhexyl)amino]carbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzoate cobaltate complexes

Cas Number:

71888-93-2

Molecular formula:

C63H66ClN10O12xCo

IUPAC Name:

Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, 4-[(5-chloro-2-hydroxyphenyl)azo]-4,5-dihydro-3-methyl-1-phenyl-3H-pyrazol-3-one 4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-1-phenyl-3H-pyrazol-3-one 3-[[1-[[(2-ethylhexyl)amino]carbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzoate cobaltate complexes

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the The test concentrations were analytically verified via HPLC-DAD in the saturated solution
test item and the control at the start of the exposure (0 hours). At the start of the exposure (0 hours), sampling was carried out
after preparation of the saturated solution. The measured concentrations were below the SysQL at the start of the
exposure (0 hours) and therefore analytical monitoring at the end of the exposure was waived and the test item
concentrations were considered to be below the SysQL in the aged media, too.

Quality criteria for the Recoveries of the test item should be within ± 20 % of the nominal or initially measured concentrations.
analytical monitoring

Sample Storage All original samples were stored at 6 ± 2 °C until sample preparation. Prepared samples were stored in an autosampler
at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the The saturated solution (1.00 mg/L test item was weighed out) wasprepared with dilution water
saturated solution two days prior to the start of the exposure (at -48 hours).

Treatment The test item was dispersed in the dilution water by stirring with approximately 1100 rpm for 48 hours at room
temperature. After completion of stirring, the test item dispersion was filtered with a membrane filter (0.45 µm, RC,
MACHEREY-NAGEL) to achieve the saturated solution. Centrifugation was found to be unsuitable.

Test concentration The effects of the saturated solution of the test item were tested in a limit test.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Clone 5
- Source:
Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin
Breeder: DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Age at study initiation (mean and range, SD): 2 to 24 hours old daphnids from a healthy stock were used for the study
- Method of breeding:
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. 20 µE x m-2 x s-1
Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
Cultur feeding: 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
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: At least 2 hours in dilution water.
- Acclimation conditions (same as test or not): Same as test conditions.
- Health during acclimation (any mortality observed): No mortality observed.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

274 mgCaCO3/L

Test temperature:

18 - 22°C, constant within ± 1°C
At start of exposure: 20.4

pH:

pH at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) of the limit concentration and the control)

Nominal loading
of the test item
[mg/L] pH-value

1.00 7.93
(Saturated solution)
Control 7.97


pH at the End of the Exposure (48 hours)
(measured in all replicates (containing daphnids))

Nominal loading
of the test item
[mg/L] pH-values
Replicates
1 2 3 4
1.00 7.75 7.76 7.76 7.75
(Saturated solution)
Control 7.59 7.70 7.74 7.75

Dissolved oxygen:

O2-concentration at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) of the limit concentration and the control)

Nomonal loading
of the test item
[mg/L]
Dissolved
O2-concentration
[mg/L]
1.00 6.22
(Saturated solution)
Control 9.01



O2-concentration at the End of the Exposure (48 hours)
(measured in all replicates (containing daphnids))

Nominaltest loading
of the test item
[mg/L] Dissolved O2 -concentration [mg/L]
Replicates
1 2 3 4
1.00 8.93 8.99 8.89 8.83
(Saturated solution
Control 8.77 8.81 8.83 8.75

Nominal and measured concentrations:

Nominal loading: 1.00 mg/L (saturated solution)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
- Test volume: 20 mL
- Aeration: None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202, Annex 3
Component Concentration [mg/L]
KCl 5.75
NaHCO3 64.8
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123

- Water Quality Parameters of the Dilution Water prior to the Start of the Exposure (0 hours):
pH-value Dissolved O2 concentration [mg/L] Temperature[°C] Conductivity[µS/cm] Total hardness [mg CaCO3/L]
7.97 9.01 20.4 670 274

- Culture medium different from test medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: test start (0 hours) and test end (48 hours)

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 20 µE x m-2 x s-1 (max. 1340 lx)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobilisation, 24 hours interval

TEST CONCENTRATIONS
-The effects of the saturated solution of the test item were tested in a limit test.

- Range finding study: No

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

other: EL10,50,100

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 2

Duration:

48 h

Dose descriptor:

other: EL10,50,100

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 3

Duration:

48 h

Dose descriptor:

other: NOEL

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 4

Duration:

48 h

Dose descriptor:

other: LOEL

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Details on results:

See below

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 hours. The EC50-value with 95 % confidence limits (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 2.00 mg/L (CI 1.76 - 2.27 mg/L)
The EC50-value of the reference item potassium dichromate after 24 hours is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Any other information on results incl. tables

Results of the Definitive Test

 

The percentage immobility, determined in the saturated solution with a nominal loading of 1.00 mg/L of the test item and the control after 24 and 48 h of exposure under static conditions, is presented in the table below. There was no effect observed in the saturated

solution and the control.

Immobilisation Rates of the Definitive Test after 24 and 48 hours of Exposure

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

Test item Nominal loading of the test item   [mg/L]	IMMOBILISATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
1.00 (Saturated solution)	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

All replicates of the saturated solution of the test item were slightly pink and visually clear throughout the exposure period.

According to OECD Guidance Document No 23 (2000) if an effect cannot be detected in a test with a saturated solution the result has to be reported as no toxic effects at saturation.Exposures are expressed in terms of the original concentration of the test itemSavinyl-Feuerrot 3GLS in water at the start of the mixing period (loading rate).  

  Measured Exposure Concentrations during the Definitive Test

 

The test concentration of the test item was analytically verified via HPLC-DAD in the saturated solution and the control only at the start of the exposure (0 hours).

The measured concentration was below the SysQL at the start of the exposure (0 hours) in the saturated solution and in the control. Therefore, analytical monitoring at the end of the exposure was waived and the test item concentrations were considered to be below the SysQL in the aged media, too.

 

Table4:      Measured Concentrations of the Test Item in the Definitive Test

Sampling date	2015-06-24 Start of the exposure, 0 hours
Date of analysis	2015-06-24*
Nominal loading of the test item [mg/L]	Test item
	Meas. conc. [mg/L]
1.00 (Saturated solution)	< SysQL
Control	< SysQL

*                    = reanalysed on 2015-06-25

Meas. conc.= measured concentration of the test item, mean value of 2 injections

SysQL            = system quantification limit of the analytical method (0.300 mg/L of the test item)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

At the saturated solution with a nominal loading of 1.00 mg/L of the test item , no effects were observed on Daphnia magna.

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the saturated solution with a nominal loading of 1.00 mg/L of the test item was determined according to OECD 202 (2004). The limit test was conducted under static conditions over a period of 48 hours.

The test item has a water solubility of < 0.03 mg/L at 20 °C. Therefore, a saturated solution with a nominal loading of 1.00 mg/L was prepared bystirring with approximately 1100 rpm for 48 hours at room temperature followed by filtration with a membrane filter (0.45 µm, RC,Macherey-Nagel) to remove undissolved test item to achieve the saturated solution.The saturated solution was slightly pink and visually clear throughout the exposure phase of 48 hours.

Twenty daphnids were exposed to the saturated solutionand the control.

The test concentrations of the test item were analytically verified via HPLC-DAD in the saturated solution and the control only at the start of the exposure (0 hours). Details of the analytical method are presented in section13.

The measured concentrations were below the SysQL at the start of the exposure (0 hours) in the saturated solution and in the control. Therefore, analytical monitoring at the end of the exposure was waived and the test item concentrations were considered to be below the SysQL in the aged media, too.The analytical results arepresented inTable4.

According to OECD Guidance Document No 23 (2000), if an effect cannot be detected in a test with a saturated solution, the result has to be reported as no toxic effects at saturation.Exposures are expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate). The EL-values are presented inTable1.

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 hours) and at the end of the exposure (48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

 

 

At the saturated solution with a nominal loading of 1.00 mg/Lof the test item , no effects were observed on Daphnia magna.

 

Table1:   EL₁₀-, EL₅₀-EL₁₀₀-Values, NOEL and LOEL

                  (based on the nominal loading levels of the test item)

Effect levels	Test duration [h]	Test item
		Nominal loading level of the test item [mg/L]
EL10 / 50 / 100	24	> 1.00
	48	> 1.00
NOEL	48	1.00
LOEL	48	> 1.00