Hydrogen bis[1-[(5-chloro-2-hydroxyphenyl)azo]-2-naphtholato(2-)]chromate(1-)

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:

08 December 2017 - 22 January 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 13, 2004

Deviations:

yes

Remarks:

methodological limitations, e.g. test conc. spacing factor was 2.5 (instead of 2.2), discrepancy between results of solubility test and actually chosen test conc., inconsistent analytical procedures

Principles of method if other than guideline:

- The highest achievable test concentration (5 mg/L) did not result in 100% immobilisation.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen bis[1-[(5-chloro-2-hydroxyphenyl)azo]-2-naphtholato(2-)]chromate(1-)

EC Number:

250-774-0

EC Name:

Hydrogen bis[1-[(5-chloro-2-hydroxyphenyl)azo]-2-naphtholato(2-)]chromate(1-)

Cas Number:

31714-55-3

Molecular formula:

C32H19Cl2CrN4O4

IUPAC Name:

[1-{[5-chloro-2-(hydroxy-kappaO)phenyl]diazenyl}-2-naphtholato(2-)-kappaO]{1-[(5-chloro-2-hydroxyphenyl)diazenyl]-2-naphtholato-kappaO}chromium

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Black powder
- Storage conditions: At room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: test samples from each replicate were drawn and pooled and mixed together for each group
- Volume: 10 mL (from each replicate)
- Frequency: at t=0 and t=48 h
- Sample storage conditions before analysis: not applicable, samples were analysed directly without storage

Test solutions

Vehicle:

yes

Remarks:

dimethyl formamide

Details on test solutions:

TEST FOR SOLUBILITY:
Before conducting the preliminary range finding study, the test item was checked for solubility and precipitation in algal medium, acetone and dimethyl formamide (DMF). Based on the results, DMF was chosen as the appropriate vehicle for the test item.

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: a quantity of 13, 32 and 80 mg test item was dissolved in 10 mL DMF to obtain the nominal concentrations of 1.3 mg/mL (stock A), 3.2 mg/mL (stock B) and 8.0 mg/mL (stock C), and a quantity of 20 and 50 mg test item was dissolved in 1 mL DMF to obtain the nominal concentrations of 20.0 mg/mL (stock D) and 50.0 mg/mL (stock E) , respectively.
Volumes of 10 µL from the stock solutions A to E and 1000 µL blank medium with 5 daphnids were taken and diluted to 100 mL with reconstituted water in respective beakers to obtain the nominal test concentrations of 0.13, 0.32, 0.80, 2.00 and 5.00 mg/L.
- Controls: for the blank control group, 1000 µL blank medium with 5 daphnids was transferred using a micropipette and diluted to 100 mL with reconstituted water. For the vehicle control group, 10 µL DMF and 1000 µL blank medium with 5 daphnids were taken and diluted to 100 mL with reconstituted water.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: daphnia
- Details on organisms: healthy daphnia were used, showing no signs of stress such as presence of males and ephippia, a delay in the production of the first brood, discolouration, etc. and having normal behavior
- Age at study initiation: <24 hours (first instar nymph)
- Source (Master culture): MicroBio Test Inc, Kleimoer 15, 9030 Mariakerke (Gent), Belgium.
- Feeding during test: no

ACCLIMATION
- Acclimation period: >48 hours
- Acclimation conditions: same as test
- Type and amount of food: living algal cells
- Health during acclimation: no mortality observed

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: first instars were collected using a micropipette

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

196 mg CaCO3/L at t=0 in the main test

Test temperature:

20.1-20.7 °C throughout the main test

pH:

7.76-7.95 throughout the main test

Dissolved oxygen:

8.74-8.93 mg/L throughout the main test

Nominal and measured concentrations:

Nominal concentrations: 0.13, 0.32, 0.8, 2.0 and 5.0 mg/L
Measured concentrations: 0.13, 0.32, 0.80, 1.93, 4.97 mg/L at t=0 and 0.12, 0.30, 0.81, 2.00 and 5.18 mg/L at t=48 h, respectively, corresponding to above stated nominal concentrations.
The measured concentrations at t=48 h were 90 - 104% of nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker with a fill volume of 600 mL
- Type: open
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per blank control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dilution water was according to OECD 202. The water was aerated prior to use so that dissolved oxygen concentration had reached saturation.
- Intervals of water quality measurement: Temperature, pH, and dissolved oxygen content (using Multi portable meter) were recorded at 0 and 48 h. Total hardness was measured once during the test period using the titrimetric method.
- Other: Mobility of the daphnids was assessed by gently swirling the test container for 15 seconds and observing their swimming behaviour. Daphnids unable to swim during the agitation of the test container and remaining settled on the water surface or settled at the bottom of the test vessel were recorded as immobile.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h dark
- Light intensity: 1320-1350 Lux

EFFECT PARAMETERS MEASURED: immobility was recorded at t=0, t=24 and t=48.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.0, 0.001, 0.01, 0.1, 1.0 and 5.0 mg/L
- Results used to determine the conditions for the definitive study: yes, the observed immobility was 30% at a test concentration of 1 mg/L and 60% at a concentration of 5.0 mg/L. The expected EC50 was between these two test concentrations and based on these result, the test concentrations for the main study were selected.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (January 2018)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 1.62-4.61 mg/L

Details on results:

- Behavioural abnormalities: no
- Other biological observations: at t=48 h, lethargy was observed at test concentrations of 0.80, 2.00 and 5.00 mg/L
- Mortality of control: no mortality occured either in the blank control or in the vehicle control
- Measured concentrations at t=48 h were 90 - 104% of nominal concentrations. Based on these results, the effect parameters were based on nominal values.
- Test conditions were within the limits prescribed by OECD guideline 202 during the exposure period.
- For details on incidence of immobilisation, see table 2 in 'any other information on results' section.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Tested concentrations: 0.09, 0.20, 0.44, 0.96 and 2.11 mg/L
- 48h-EC50: 0.24 mg/L

Reported statistics and error estimates:

The 48 h EC50 and the associated 95% confidence limits were calculated following the Probit analysis method (Finney, 1971) using in-house developed, validated software.

Any other information on results incl. tables

Table 1 Measured concentrations of T-95/T-99 (main study)

Groups	Nominal Conc. based on Purity of Test Item (mg/L)	t=0 h			t=48 h
		Peak Area of Sample (Y)	Analysed  Conc. (mg/L)	Recovery^# (%)	Peak Area of Sample (Y)	Analysed Conc. (mg/L)	Recovery^# (%)
G1 (0.00)	0.00000	ND	0.00000	0.00	ND	0.00000	0.00
G2 (0.00)	0.00000	ND	0.00000	0.00	ND	0.00000	0.00
G3 (0.13)	0.12922	11518	0.13228	102.37	8875	0.11674	90.34
G4 (0.32)	0.31808	27943	0.31881	100.23	24993	0.30040	94.44
G5 (0.80)	0.79520	70654	0.80385	101.09	69301	0.80527	101.27
G6 (2.00)	1.98800	169559	1.92704	96.93	174237	2.00097	100.65
G7 (5.00)	4.97000	87373	4.96858	99.97	89605	5.18313	104.29
Intercept of Y-axis (a)		-130.58			-1370.45
Slope of the line (b)		88056.95			87761.04

ND = Not Detected, Conc. = Concentration.

^# relative to nominal

Dilution factors for the samples of groups G1, G2, G3, G4, G5, G6 and G7 were 1.0, 1.0, 1.0, 1.0, 1.0, 1.0 and 5.0, respectively.

Table 2 Incidence of immobility (main study)

Group	Nominal Test Concentration (mg/L)	N° of Replicates	N° of Daphnids/ group	Immobility N° and % at
				0 h		24 h		48 h
				N°	%	N°	%	N°	%
G1	Control (0.0)	4	20	0	0	0	0	0	0
G2	Vehicle control (0.0)	4	20	0	0	0	0	0	0
G3	0.13	4	20	0	0	0	0	0	0
G4	0.32	4	20	0	0	0	0	1	5
G5	0.80	4	20	0	0	0	0	5	25
G6	2.00	4	20	0	0	0	0	8	40
G7	5.00	4	20	0	0	0	0	13	65

h = Hour

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'overall remarks' section

Conclusions:

The 48h-EC50 of T95/T-99 towards Daphnia magna was determined to be 2.73 mg/L (95% CI: 1.62-4.61 mg/L).

Executive summary:

In an short-term toxicity test performed according to OECD 202 and GLP principles, T-95/T-99 was assessed for its effect on Daphnia magna.

The test item was initially tested for solubility and dimethyl formamide was chosen as the most appropriate vehicle.

Based on a preliminary test, nominal concentrations of 0.13, 0.32, 0.8, 2.0 and 5.0 mg/L were chosen for the main test and both a blank control and a vehicle control were included. The test period was 48 hours, under static conditions, in which immobility was recorded at t=24 and t=48 h. Test samples for analysis of test concentrations were taken at t=0 and t=48 h. Results showed that the test item concnetrations were in the range of 90.3 -104.3% of nominal concentrations throughout the test period, and 88.2 -104.3% of initial measured concentrations at the end of the test. Based on these results, effect parameters were based on nominal concentrations.

The 48h-EC50 of T-95/T-99 towards Daphnia magna was determined to be 2.73 mg/L.