Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

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:

July 19, 2016 to September 05, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline; EU Method; GLP

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 202 (Daphnia sp. Acute Immobilisation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

EC Number:

276-423-1

EC Name:

Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

Cas Number:

72152-71-7

Molecular formula:

C16H8CuN2O8S2.2Na

IUPAC Name:

Cuprate(2-), [6-(hydroxy-kO)-5-[2-[2-(hydroxy-kO)-5-sulfophenyl]diazenyl-kN1]-2-naphthalenesulfonato(4-)]-, sodium

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control were collected at the end of the test (after 48 hours) by pouring together the contents of the test beakers of each treatment.
Additional samples of the control were taken

Test solutions

Vehicle:

no

Details on test solutions:

The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 80.4 mg test item into 804 mL test water by intense stirring for 10 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media.
The test media were prepared just before introduction of the Daphnia (= start of the test).

Test Concentrations: 100, 45.5, 20.7, 9.4 and 4.3 mg test item/L and a control

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 4.25 to 21.75 hours old
Sex: Female
Origin: The Daphnia introduced in the test were taken from ibacon's in house laboratory culture.
Breeding Conditions: The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental Daphnia was performed in Elendt M4 medium (see 6.5). The test organisms were not first brood progeny. The Daphnia in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Water Hardness: 2.5 mmol/L (= 250 mg/L) as Calcium Carbonate

Test temperature:

21 °C at test start;
21 °C at test end

pH:

8.2 at test start;
8.1 at test end; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

8.8 to 8.9 mg/L at test start;
8.6 to 8.7 mg/L at test end

Nominal and measured concentrations:

100, 45.5, 20.7, 9.4 and 4.3 mg test item/L and a control

Details on test conditions:

Test Environment: Controlled environment room
Measurement of pH, Dissolved
Oxygen and Water Temperature:
The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.
Water Temperature: 21 °C at test start;
21 °C at test end
pH-Values: 8.2 at test start;
8.1 at test end; and thus the pH-value did not vary by more than 1.5 units
Dissolved Oxygen Concentration: 8.8 to 8.9 mg/L at test start;
8.6 to 8.7 mg/L at test end
Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 550 to 830 lux (measured once during the test).

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

45.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item concentration of 45.5 mg test item/L. At the concentration of 100 mg test item/L, six animals were immobile.

Results with reference substance (positive control):

In the most recent test with the reference item potassium dichromate the EC50 after 24 hours was determined to be 1.58 mg test item/L, indicating that the sensitivity of the Daphnia was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).

Reported statistics and error estimates:

Statistical Analysis: The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by probit analysis.
The NOEC and LOEC after 24 and 48 hours were determined directly from the raw data.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Control Immobilisation Rate was 0 % and furthermore no daphnid showed signs of disease or stress. Dissolved Oxygen Concentration was above or equal 8.6 mg O2/L in the control and test vessels at the end of the test.

Conclusions:

The toxic effect of the test item to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEC was determined to be 45.5 mg test item/L. The 48-hour LOEC was determined to be 100 mg test item/L and the 48-hour EC50 value was calculated to be > 100 mg test item/L. The 48-hour EC10 was calculated to be 86.7 mg test item/L.

Executive summary:

Title:	Acute Toxicity to Daphnia magna in a Static 48-hour Immobilisation Test
Purpose:	The purpose of this study was to determine the influence of the test item on the mobility of Daphnia magna. Young Daphnia (< 24 hours old) were exposed in a static test to the test item for 48 hours, added to test water at a range of concentrations. Under otherwise identical test conditions, different concentrations of the test item result in different percentages of Daphnia being no longer capable of swimming at the end of the test. The test method of application and the test species Daphnia magna are recommended by the test guidelines. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.
Guidelines:	-     Commission Regulation (EC) No 440/2008, Annex, Part C, C.2.: “Daphnia sp. Acute Immobilisation Test”, Official Journal of the European Union (EN), dated May 30, 2008 -     OECD Guideline for Testing of Chemicals No. 202: “Daphnia sp., Acute Immobilisation Test” adopted April 13, 2004 -     SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
Material and Methods
Test Item:	batch no.:101115; purity: 92.1% w/w , according to certificate of analysis
Test Species:	Daphnia magna, clone 5;4.25to 21.75 hoursold Source: The Daphnia introduced in the test were taken from ibacon’s in-house laboratory culture.
Test Design:	This study encompassed 6 treatment groups (5 dose rates of the test item and a control) each containing 20 individuals. The mobility of the daphnia was determined in a static 48-hour test by visual observation after 24 and 48 hours.
Endpoints:	Number of immobile organisms after 24 and 48 hours
Test Concentrations:	100, 45.5, 20.7, 9.4 and 4.3 mg test item/L and a control
Test Conditions:	Water temperature:21 °C; pH value:8.1to 8.2; dissolved oxygen concentration:8.6to 8.9mg/L; photoperiod: 16 h light – 8 h dark; light intensity:550 to 830 lux; and thus were within the ranges requested by guideline OECD 202
Results
Biological test results:	After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item concentration of 45.5mg test item/L. At the concentration of 100mg test item/L, six animals were immobile.
	Table1. Summary of Biological Results
	Nominal Concentration	% of immobilised Daphnia after
	[mg test item/L]	24 hours	48 hours
	Control	0	0
	4.3	0	0
	9.4	0	0
	20.7	0	0
	45.5	0	0
	100	20	30
	EC50[mg/L]:	> 100	> 100
	95 % CI [mg/L]:	n.d.	n.d.
	EC20[mg/L]:	100	94.2
	95 % CI [mg/L]:	n.d.	n.d.
	EC10[mg/L]:	91.5	86.7
	95 % CI [mg/L]:	n.d.	n.d.
	NOEC [mg/L]:	45.5	45.5
	LOEC [mg/L]:	100	100
	Values refer to nominal test concentrations CI: Confidence interval n.d.: not determinable NOEC and LOEC values determined directly from raw data
Analytical Results:	The quantification of the test item in the test samples was performed using liquid chromatography with UV detection. At the start of the test 100 % of the nominal test concentrations were found (average of all test concentrations). After 48 hours test duration, 97 % of the nominal value was determined (average of all test concentrations). During the test the daphnids were exposed to a mean of 98 % of nominal. Therefore, all reported results refer to nominal concentrations.
Conclusion:	The toxic effect of the test item to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEC was determined to be45.5 mg test item/L. The 48-hour LOEC was determined to be 100 mg test item/Land the 48-hour EC50value was calculated to be> 100 mg test item/L. The 48-hour EC10was calculated to be 86.7 mg test item/L.

 

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.