Reaction products of 6-amino-4-hydroxynaphthalene-2-sulphonic acid and 7-amino-4-hydroxynaphthalene-2-sulphonic acid, condensed with Formaldehyde and Sodium hydrogensulfite, coupled with diazotised Sodium 2-amino-4,6-dinitrophenoxide, metalized with Sodium Dichromate, sodium salts

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:

04 June - 06 June, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

Experimental temperature 20.0 – 23.8 °C [not in the desired range (18.0 – 22.0°C)]. Experimental temperature f 20.0 – 23.8 °C [fluctuated by more than ± 1 °C]. As no immobility was observed in the blank control these deviations was stated as uncritical.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

see report attached

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing 100.6 mg/L test item in dilution water was prepared. The lower treatments (4.6 / 10 / 22 / 46 mg/L nominal) were prepared by dilution of this stock solution with dilution water. The appearance of the final solutions was clear and free of turbidity.
The concentrations 46 mg/L (2x) and 100 mg/L (5x) were diluted, because they were not in the calibrated range.
- Controls:
Blank control 4 vessels, each containing 20 ± 5 mL dilution water and 5 daphnia
- Chemical name of vehicle: Water
- Concentration of vehicle in test medium:
stock solution containing 100.6 mg/L test item
final test solutions 4.6 / 10 / 22 / 46 mg/L nominal
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): the final solutions were clear and free of turbidity

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: STRAUS
- Age at study initiation (mean and range, SD): between 0 and 24 hours
- Method of breeding: In-house breeding
- Feeding during test
- Food type: green algae (Desmodesmus subspicatus)
- Photo period: 16/8 hours, using neon tubes
- Medium: M4-Medium (recipe of ELENDT)
- Medium renewal: twice a week

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

Observation times 24 and 48 hours

Test temperature:

20.0 – 23.8 °C

pH:

7.5 - 7.8

Dissolved oxygen:

8.7 - 8.9

Nominal and measured concentrations:

4.6 / 10 / 22 / 46 / 100 mg/L (nominal concentration)
4.09 / 9.59 / 22.84 / 47.20 / 11.46 mg/l (measured concentration t= 48h)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7

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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

FINDINGS

Immobility

In the blank control, none of the daphnia was immobilised (see table below).

Table: Immobility

Conc (mg/l)	Immobility 24 hours						Immobility 48 hours
	Absolute			%			Absolute			%
Blank	0	0	0		0	0	0	0	0		0	0
4.6	0	0	0		0	0	1	0	0		0	5
10	0	0	0		0	0	0	0	0		0	0
22	0	0	0		0	0	0	0	0		0	0
46	0	0	0		0	0	0	1	0		0	5
100	0	0	0		1	5	0	3	2		2	35

 

pH and O2

The pH values and the concentration of dissolved oxygen in the test media and the blank

control are given in the following table:

Table: pH and O2-values

Conc (mg/l)	pH		O2-Concentration in mg/L
	0 h	48 h	0 h	48 h
Blank	7.8	7.7	8.9	8.7
4.6	7.8	7.7	8.9	8.7
10	7.8	7.7	8.9	8.8
22	7.8	7.7	8.9	8.7
46	7.7	7.7	8.9	8.7
100	7.5	7.7	8.9	8.7

 

Analytical Determinations

At the beginning and at the end of the test, the content of the test item in the test solutions was determined using a photometer. The concentrations determined at the start of the test laid between 82 % and 101 % of the nominal concentration. At the end of the test the determined concentrations laid between 89 % and 111 % of the nominal concentration.

Therefore, the determination of the biological results was based on the nominal concentration (see Guidance Doc. No.23 (§176)). The measured QC sample (10 mg/L) was taken into account at the beginning (96.7 %) and the end of the test (96.4 %). The concentrations 46 mg/L (2x) and 100 mg/L (5x) were diluted, because they were not in the calibrated range.

The measured concentrations for treatment and blank control are given in the following table:

 

Table: Raw Data and Measured Concentration

 

Nominal concentration	Absorbance t = 0 h	Absorbance t = 48 h	Meas. Conc. T= 0 h*	Meas. Conc. T= 48 h*
			mg/l	mg/l
Blank control	0.0000	0.0000	0.016	0.16
	0.0000	0.0001	0.016	0.025
4.6	0.0387	0.0419	3.640	3.940
	0.386	0.0419	3.630	3.940
10	0.0926	0.0982	8.715	9.245
	0.0925	0.0982	8.706	9.245
22	0.2147	0.2321	20.341	22.013
	0.2147	0.2321	20.341	22.013
46	0.2357	0.2398	22.359	22.754
	0.2356	0.2397	22.350	22.744
100	0.261	0.2267	19.517	21.494
	0.2060	0.2266	19.507	21.484
QC (10 mg/l)	0.1027	0.1024	9.670	9.642
	0.1027	0.1023	9.670	9.632

*Without dilution factor and recovery of the QC samples

 

Table: Measured Concentrations and % of Nominal Concentration

 

Nominal conc. (mg/l)	Meas. Conc t = 0 h (mg/l)	Meas. Conc t = 48 h (mg/l)	% of Nominal Conc. t = 0 h	% of Nominal Conc. t = 48 h
Blank	0.02	0.02	-	-
4.6	3.76	4.09	82	89
10	9.01	9.59	90	96
22	21.04	22.84	96	104
46	46.24	47.20	101	103
100	100.89	111.46	101	111
LOQ	0.5	0.5	-	-

LOQ = limit of quantification

 

RESULTS AND STATISTICS

The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.3.0.

 

VALIDITY

-         Immobilisation in the controls may not exceed 10 %.

Immobilisation in the controls was 0 %.

-         The concentration of dissolved oxygen at the end of the test must be at least 3 mg/L.

The lowest concentration of dissolved oxygen at the end of the test was 8.7 mg/L.

Validity criteria fulfilled:

yes

Remarks:

Immobilisation in the controls may not exceed 10 % [It was 0 %]. The concentration of dissolved oxygen at the end of the test must be at least 3 mg/L [It was 8.7 mg/L].

Conclusions:

48h-EC50 > 100 mg/L

Executive summary:

Guidelines

One valid experiment was performed according to the OECD Guideline 202.

 

Discussion

The study was performed using 5 concentrations ranging from 4.6 to 100 mg/L nominal concentration. For each test concentration and the blank control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted. Three concentrations showed toxicity between 5 and 35 % immobilisation. None of the animals was immobilised in the blank control. Because of the immobilisation of 35 % in the highest tested concentration at 48 h, the 48h-EC50was set as higher than the highest tested concentration. At the beginning and at the end of the test, the content of the test item in the test solutions was determined using a photometer. The concentrations determined at the start of the test laid between 82 % and 101 % of the nominal concentration. At the end of the test the determined concentrations laid between 89 % and 111 % of the nominal concentration. Therefore, the determination of the biological results was based on the nominal concentration. Potassium dichromate K2Cr2O7(CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable. No observations were made which might cause doubts concerning the validity of the study outcome. All validity criteria were met.

The result of the test is considered valid.

 

Results

The following results were determined for the test item (species:Daphnia magna).

48h-NOEC = 46 mg/L

48h-LOEC = 100 mg/L

24h-EC50 > 100 mg/L

48h-EC50 > 100 mg/L

Description of key information

EC50 (48h) > 100 mg/l

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

The acute toxicity of the test item to aquatic invertebrated was investigated on Daphnia magna STRAUS according to the OECD Guideline 202.

the test was performed using 5 concentrations ranging from 4.6 to 100 mg/L nominal concentration. For each test concentration and the blank control, 20 daphnia were exposed to the test item for 48 hours in a static test system.

After 24 and 48 hours, the immobilised daphnia were counted. None of the animals was immobilised in the blank control. Because of the immobilisation of 35 % in the highest tested concentration at 48 h, the 48h-EC50 was set as higher than the highest tested concentration (EC50 48h >100 mg/l)