Reaction products of diazotised 2-amino-5-nitrobenzenesulfonic acid coupled with resorcinol, subsequently coupled with diazotized 2-amino-4-nitro-6-sulfo-phenol, chelated with iron, sodium salt

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 9 to 20, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations analysed in preliminary test: 5 and 100 mg/l
- Concentrations analysed in definitive test: 21 and 100 mg/l
- Time: beginning and end of the test. Samples for analysis (0 hours) were prepared and immediately delivered in transport box to analytical laboratory; samples were analysed on the day of delivery. Samples for analysis (48 hours) were delivered to analytical laboratory immediately after the end of testing; samples were analysed on the day of delivery.
- Storage: all samples were stored at laboratory temperature.

Vehicle:

no

Details on test solutions:

Stock solution of test substance was prepared in dilution water. 50 mg of test substance was weighed into 500 ml of dilution water for the preliminary test and 100 mg of test substance was weighed into 1000 ml of dilution water for the definitive test. The stock solution was ultrasonicated for 5 minutes.
Concentrations of solutions used in the preliminary and definitive test were obtained by dilution of the stock solution with dilution water.

Preliminary test
The stock solution of the test item in dilution water was prepared for the preliminary test. The test was performed in a range of nominal concentrations from 1 mg/l to 100 mg/l. Test solutions were prepared by dosing of appropriate volumes of the stock solution into volumetric flasks and replenishment up to mark with dilution water. Control test was performed concurrently with dilution water only. The test was performed in four replicates. The volume of the test solution was 50 ml in each test beaker. 5 animals were put into each beaker. The daphnia immobilisation was observed and recorded after 24 and 48 hours. At the beginning and at the end of the test, ambient temperature, the oxygen content and pH value were measured. The conductivity of deionised water and content of Ca and Mg ions in dilution water were measeured at the beginnign of the test. The test was performed as static.

Definitive test
Test solutions were prepared by dosing of appropriate volumes of stock solution into volumetric flasks and replenishment up to mark with dilution water. The geometric concentration series of test substance with factor 1.6 was used for the test. Control test was carried out concurrently with dilution water only. The test was performed in four replicates. The volume of test solution was 50 ml in each test beaker. 5 animals were put into each beaker. Total number of daphnia used for each test concentration and control was 20. The daphnia immobilisation was observed after 24 and 48 hours. At the beginning and at the end of the test, ambient temperature, oxygen content and pH value were measured. The test was performed as static.
The stock solution of the test item in dilution water was prepared for the definitive test. The definitive test was performed in a range of nominal concentrations from 21 mg/l to 100 mg/l. The test solutions were prepared by dosing of appropriate volumes of stock solution into volumetric flasks and replenishment up to mark with dilution water. The geometric concentration series of the test item with factor 1.3 was used for the test. Control test was carried out concurrently with dilution water only. The test was performed in four replicates. The volume of the test solution was 50 ml in each test beaker. 5 animals were put into each beaker. Total number of daphnia used for each test concentration and control was 20. The daphnia immobilisation was observed and recorded after 24 and 48 hours. At the beginning and at the end of the test, the ambient temperature, the oxygen content and pH value were measured. The conductivity of deionized water and content of Ca and Mg ions in dilution water were measured at the beginning of the test.
The test was performed as static.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Feeding during test: no
- Holding and culturing: M4 medium
- Test medium: dilution water (according to Annex 2 of EU method C.2)

ACCLIMATION
- Acclimation period: 48 h prior to start of the test
- Acclimation conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Preliminary test: 2.54 mmol/l in dilution water - Definitive test: 2.52 mmol/l in dilution water.

Test temperature:

Preliminary test: 22 - 20.4 °C
Definitive test: 20.7 - 20.3 °C
Measured at start and end of test.

pH:

Preliminary test: 7.7 and 8.0
Definitive test: 7.5 and 7.8
Measured at start and end of test.

Dissolved oxygen:

Preliminary test: 7.4 - 7.7 mg/l
Definitive test: 7.6 - 8.2 mg/l
Measured at start and end of test.

Conductivity:

Preliminary test: 1.18 µS/cm - Definitive test: 1.60 µS/cm.

Nominal and measured concentrations:

Nominal concentrations - preliminary test: 100, 50, 10, 5, 1 mg/l and control - Nominal concentrations - definitive test: 100, 77, 59, 45, 35, 27, 21 mg/l and control.

Details on test conditions:

TEST SYSTEM
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
Dilution water was prepared just before the test by admixing 2.5 ml of stored solutions A, B, C and D into 1000 ml of deionised water.

A. 117.6 g CaCl2×2H20 in 1 l deionised water
B. 49.3 g MgSO4×7H2O in 1 l deionised water
C. 25.9 g NaHCO3 in 1 l deionised water
D. 2.3 g KCl in 1 l deionised water

EFFECT PARAMETERS MEASURED: immobilisation after 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: 100, 50, 10, 5, 1 mg/l
- Results used to determine the conditions for the definitive study: yes

QUALITY CRITERIA
1. immobilisation in the controls must not exceed 10 % at the end of the test.
2. dissolved oxygen concentrations (throughout the test) should remain above 3 mg/l.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

84.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Probit analysis

Details on results:

QUALITY CRITERIA
1. immobilisation in the control was 0 %.
2. the lowest value of dissolved oxygen concentration measured in the study was 7.4 mg/l.

EC50 (24h) > 100 mg/l
EC50 (48h) = 84.2 mg/l (95 % confidence limit: 70.3 – 114.1 mg/l)
EC0 (24h) = 59 mg/l
EC0 (48h) = 27 mg/l
EC100 (24h) > 100 mg/l
EC100 (48h) > 100 mg/l

Results with reference substance (positive control):

EC50 (24h) = 1.10 mg/l
EC50 (48h) = 0.77 mg/l

Immobilisation of daphnia - preliminary test

time	replicate	concentration
		100	50	10	5	1	control
mobile animals at 24 h	1	5	5	5	5	5	5
	2	5	5	5	5	5	5
	3	5	5	5	5	5	5
	4	5	5	5	5	5	5
	total	20	20	20	20	20	20
% immobilisation at 24 h		0	0	0	0	0	0
mobile animals at 48 h	1	2	2	5	5	5	5
	2	2	2	5	5	5	5
	3	3	5	5	5	5	5
	4	1	1	5	5	5	5
	total	8	10	20	20	20	20
% immobilisation at 48 h		60	50	0	0	0	0

Concentrations - preliminary test

nominal conc. mg/l	determined conc. 0 h mg/l	determined conc. 48 h mg/l	change of test item conc. in interval 0-48 h (% of initial conc.)
5	4.2	5.3	+ 26.2 %
100	90.6	78.7	- 13.1 %

Immobilisation of daphnia - definitive test

time	replicate	concentration
		100	77	59	45	35	27	21	control
mobile animals at 24 h	1	5	5	5	5	5	5	5	5
	2	4	5	5	5	5	5	5	5
	3	5	4	5	5	5	5	5	5
	4	5	5	5	5	5	5	5	5
	total	19	19	20	20	20	20	20	20
% immobilisation at 24 h		5	5	0	0	0	0	0	0
mobile animals at 48 h	1	2	2	4	3	4	5	5	5
	2	2	3	3	4	5	5	5	5
	3	2	3	4	4	3	5	5	5
	4	3	2	4	5	5	5	5	5
	total	9	10	15	16	17	20	20	20
% immobilisation at 48 h		55	50	25	20	15	0	0	0

Concentrations - definitive test

nominal conc. mg/l	determined conc. 0 h mg/l	determined conc. 48 h mg/l	change of test item conc. in interval 0-48 h (% of initial conc.)
21	20.3	25.5	+ 25.6 %
100	99.2	125.4	+26.4 %

Validity criteria fulfilled:

yes

Conclusions:

EC0 (48h) = 27 mg/l
EC50 (48h) = 84.2 mg/l
EC100 (48h) > 100 mg/l

Executive summary:

Method:

A study on daphnia magna was carried out according to OECD guideline 202 under static exposure conditions.

A preliminary test was performed at nominal concentrations of 100, 50, 10, 5, 1 mg/l and control. Concentrations used in main test were chosen on the basis of preliminary results and were: 100, 77, 59, 45, 35, 27, 21 mg/l and control. In both preliminary and main test, exposure duration was 48 hours; observations for effects on mobility were done at 24 and 48 h after start of exposure. Tests were run using 20 daphnids per concentration divided into 4 replicates.

Analytical monitoring of test concentrations was done at 5 and 100 mg/l in the preliminary test and at 21 and 100 mg/l in the main test, at 0 h and 48 h after start.

 

Results:

Analytical results showed that the substance concentration varied slightly more than ± 20 %. As the increase was only ca. 6 % above the limit, nominal concentrations were also used for all evaluations.

Effects on mobility were:

EC₅₀ (24h) > 100 mg/L.

EC₅₀ (48h) = 84.2 mg/L (95 % confidence limit: 70.3 – 114.1 mg/L).

EC₀ (48h) = 27 mg/L.

EC₁₀₀ (48h) > 100 mg/L.

Description of key information

EC₅₀ (48h) = 84.2 mg/L.

EC₀ (48h) = 27 mg/L.

EC₁₀₀ (48h) > 100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

84.2 mg/L

Additional information

Acute toxicity to aquatic invertebrates was assessed in a 48 -hour study under static exposure conditons, following OECD guideline 202.

In a preliminary test at concentrations between 1 and 100 mg/l, concentration-related effects on mobility were seen. Based on such results, a main test was conducted at concentrations between 21 and 100 mg/l. Controls were run and were valid. All evaluations were based on nominal concentrations, as analytical monitoring showed that concentration of test substance varied only slightly more than ± 20 % of the nominal value.

Effect levels were determined at 24 h and 48 h after start of the study.