Disodium [6-[(2-anilino-1-naphthyl)azo]-2,4-dinitrophenolato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)]chromate(2-)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

November from 07th to 09th, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Details on the read-across are available in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling method: all test concentrations and control were analytically verified at the start (0 h) and at the end of exposure (48 h). Sampling at the start of exposure (0 h) from freshly prepared media was carried out immediately after preparation of test concentrations. Samples at the end of exposure (48 h) were taken directly from additional replicates prepared with test media, but without daphnids, incubated under test conditions until sampling.
- Sample storage conditions before analysis: all original samples were stored at 6 ± 2 °C until sample preparation. Prepared samples were stored at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: a stock solution (100 mg/l of the test item were weighed out) was freshly prepared with dilution water.
- Eluate: dilution water
- Differential loading: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/l (dilution factor 2).
- Controls: dilution water without test item tested under the same conditions as test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Organism: Daphnia magna (STRAUS), clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding.
- Age at study initiation: 2 to 24 h
- Method of breeding: in 2-3 litres glass vessels with appr. 1.8 l culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µExm-2 x s-1.
- Feeding during test: no feeding.

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions: same as test.
- Health during acclimation: healthy, no mortality observed.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Test temperature:

20.5 °C

pH:

Control: 7.68 - 7.87
Test vessels: 7.95 - 8.14

Dissolved oxygen:

Control: 8.33 - 8.86
Test vessels: 8.16 - 8.82

Nominal and measured concentrations:

6.25, 12.5, 25.0, 50.0 and 100 mg/l, nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers (4 cm ID × 7 cm H), 50 ml.
- Type: closed with parafilm.
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4 replicates
- No. of vessels per control: 4 replicates

TEST MEDIUM / WATER PARAMETERS
- Dilution water: ISO test water, according to OECD 202, Annex 3: KCl 5.76 mg/l, NaHCO3 64.8 mg/l, CaCl2 × 2 H2O 294 mg/l, MgSO4 × 7 H2O 123 mg/l.
- pH-value: 7.8 ± 0.2
- Hardness: 258 mg CaCO3/l
- Conductivity: Dilution water at test start:654 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/l
- Intervals of water quality measurement: at start and end of test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16:8 light:dark cycle
- Light intensity: diffuse light, illumination strength max. 20 µE × m-2 × s-1

EFFECT PARAMETERS MEASURED
Immobility was determined in all groups after 24 h and 48 h.

TEST CONCENTRATIONS
- Range finding study: immobilization rates in the non GLP Preliminary Range Finding Test (n = 20, divided into 2 replicates with 10 daphnids each).
- Concentrations: 1, 10 and 100 mg/l, nominal.
- Results: no immobilization was recorded at none of the concentrations tested.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

30.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

EC100-values after 24 and 48 h and the NOEC and LOEC after 48 h were deduced directly from the observation data, without mathematical calculation.
All test item solutions had a concentration-related intense black colour throughout exposure. The control medium was colourless and clear.

MEASURED CONCENTRATIONS
The measured concentrations of the test item at the start of the exposure (0 h) were in the range of 93 to 102 % of the nominal values. The measured concentrations of the test item at the end of the exposure (48 h) were in the range of 84 to 97 % of the nominal values, indicating that the concentrations of the test item were successfully maintained for the duration of the test.

Results with reference substance (positive control):

EC50/LC50 (48h): 1.73 (CI 1.67 - 1.79) mg/l

Reported statistics and error estimates:

The EC10-values and the EC50-value after 24 h were calculated by sigmoidal dose-response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5, wherever appropriate. The EC50-value after 24 h was calculated as geometric mean of the highest concentration causing 0 % immobilisation and the lowest concentration causing 100 % immobilisation, because only 0 and 100 % immobilisation was observed within the tested concentration range. These concentration levels were also used as 95 % confidence limits. The concentration effect relationships are presented graphically.

Summary of the study outcomes

Endpoint	Test dutation	Nominal concentration of the test item [mg/l]	95 % confidence limits [mg/l]
EC10	24 hrs	69.2	n.a.
	48 hrs	16.4	13.2 - 19.0
EC50	24 hrs	70.7	50.0 - 100
	48 hrs	30.2	27.1 - 33.7
EC100	24 hrs	100	n.a.
	48 hrs	100	n.a.
NOEC	48 hrs	12.5	n.a.
LOEC	48 hrs	25.0	n.a.

n.a.: not applicable

Immobilization Rates in the Definitive Test after 24 and 48 h of Exposure

Nominal concentration of the test item [mg/l]	% immobilization
	24 hrs					48 hrs
	Rep. 1	Rep. 2	Rep. 3	Rep. 4	Mean	Rep. 1	Rep. 2	Rep. 3	Rep. 4	Mean
100	100	100	100	100	100	100	100	100	100	100
50	0	0	0	0	0	100	80	80	80	85
25	0	0	0	0	0	40	60	20	20	35
12.5	0	0	0	0	0	0	0	0	0	0
6.25	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Measured concentrations

Nominal concentration of the test item [mg/l]	Start		End (48hrs)
	Meas. conc. [mg/l]	% of nominal	Meas. conc. [mg/l]	% of nominal
100	102	102	96.6	97
50	50.4	101	42.2	84
25	25	100	24.2	97
12.5	12.3	98	12.1	97
6.25	5.83	98	5.67	91
Control	< LOQM		< LOQM

Meas. conc.: measured concentration of the test item, mean value of 2 injections, dilution factor taken into account

LOQM: Limit of quantification of the analytical method (1.50 mg/l of the test item)

Oxygen and pH conditions

Nominal concentration of the test item [mg/l]	pH-Value
	Start	End
100	8.11	7.96 - 7.97
50	8.08	7.96
25	8.1	7.95 - 7.96
12.5	8.11	7.97 - 7.99
6.25	8.14	7.91 - 7.98
Control	7.96	7.68 - 7.87
	Dissolved O2-Concentration
100	8.82	8.59 - 8.65
50	8.81	8.53 - 8.59
25	8.82	8.18 - 8.34
12.5	8.8	8.16 - 8.37
6.25	8.76	8.32 - 8.39
Control	8.86	8.33 - 8.43

Validity criteria fulfilled:

yes

Conclusions:

EC50 (48h): 30.2 mg/l (nominal)

Executive summary:

In an acute immobilization test with Daphnia magna (STRAUS), the effects of test item were determined according to OECD 202 (2004). The study was conducted under static conditions over a period of 48 h with 5 concentration levels of test item in the range of 6.25 to 100 mg/l, prepared with dilution water in a geometric series with a factor of 2. Twenty daphnids were exposed to each concentration level and control. Test item concentrations were analytically verified by HPLC-DAD at the start of the exposure (0 h) and at the end of the exposure (48 h) of all concentration levels and the control. Measured concentrations at start of exposure (0 h) were in the range of 93 to 102 % of nominal values; at the end of exposure (48 h), concentrations were in the range of 84 to 97 % of nominal values, indicating that concentrations of test item were successfully maintained for the duration of the test. The No Observed Effect Concentration after 48 hours was determined to be 12.5 mg/l.

Conclusion

EC50 (48h): 30.2 mg/l (nominal)

Description of key information

EC50 (48h): 30.2 mg/l (nominal)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

30.2 mg/L

Additional information

There are no information about the short-term potential toxicity of Acid Black 222 to aquatic invertebrates, thus available information on the structural analogue Similar Substance 01 have been taken into consideration; the read across approach can be considered as appropriate and suitable to assess the property under investigation. Details are available in section 13.

The effects of the Similar Substance 01 were determined on daphnids, according to OECD guideline 202 (2004). Concentrations of test item were analytically verified by HPLC-DAD: measured concentrations at the start of the exposure (0 h) were in the range of 93 to 102 % of nominal values; at the end of the exposure (48 h), concentrations were in the range of 84 to 97 % of nominal values, indicating that concentrations of test item were successfully maintained for the duration of the test. The No Observed Effect Concentration after 48 hours was determined to be 12.5 mg/l. The EC50 (48h) value was determined to be 30.2 mg/l (nominal).

A second experiment is available on Similar Substance 01. Acute toxicity of test item to Daphnia magna was determined in a 48-hour static test, according to the OECD guideline 202 and EEC Guideline C 2. The nominal concentrations tested were 10, 18, 32, 58, 100 and 180 mg test article/l; during the test, concentrations in the analysed test media were in the range of 100 - 109 % of the nominal values. No immobilization and abnormal behaviour of daphnids was observed in the control. The NOEC and EC0 values were determined to be 10 mg/l; the EC100 value was 192 mg/l after 48 hours.