Phenol, 4-[[4-(dimethylamino)phenyl]azo]-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

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:

13 February - 24 May 2018

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)

Version / remarks:

13th April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

April 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000

Version / remarks:

September 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Expiry date: 01 December 2020

Analytical monitoring:

yes

Details on sampling:

ANALYSIS OF THE SAMPLES
For determination of the test item concentration, samples were taken in duplicates from the testing concentration and from the control at the start and end of each renewal period of the test. The first series of the samples taken at the start and at the end of each renewal were sent to individual analysis. The further samples taken were kept separately as a reserve until the date of the issue of the final report at a temperature below – 15 °C.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: As the test item is poorly soluble in deionized water as well in the test medium, preparation of test item solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23). Appropriate amount of test item was suspended in the dilution water (ISO medium; see 4.4) in order to give the loading rate of 100 mg test item/L. The solution was handled by ultrasonic bath for 10 minutes thereafter stirred for a period of approximately 24 hours to achieve equilibrated concentration. The solution was then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material.
- Controls: Negative control (ISO medium without addition of test item ), positive control (with the reference substance potassium dichromate)

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: Laboraty culture; originally obtained from Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100-Gödöllő, Kotlán S. u. 3, Hungary
- Sex: Female
- Age of parental stock: Less than 24 h old at the beginning of the test.
- Feeding during test: Test animals were not fed during the exposure

ACCLIMATION
- Acclimation period: Test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation before the test was not necessary
- Acclimation conditions: Same as test
- Type and amount of food: The Daphnia were fed with concentrated algal suspension of Raphidocelis subcapitata during holding.
- Health during acclimation: Apparently healthy animals were used in this test with a known history (breeding method, pre-treatment).

Test type:

semi-static

Water media type:

other: Reconstituted water (ISO Medium, according to OECD 202)

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Animals were exposed to the test concentration or included as control over a period of 48 hours.Daphnia were observed for immobility or mortality by visual observation after 24 and 48 hours of exposure.

Post exposure observation period:

Daphnia were observed for immobility or mortality by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile.

Hardness:

231.4 mg/L (as CaCO3)

Test temperature:

The test temperature was in the range of 20.1 - 20.6°C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 19.9 – 20.8°C.

pH:

The pH was in the range of 7.83 - 8.18 during the test.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 7.47 - 8.65 mg/L during the test.

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Type: open
- Material, size: Glass, volume app. 50 mL
- Aeration: No
- Renewal rate of test solution: test solution was renewed once during the test (on day 1)
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM
- Preparation of dilution water: ISO Medium according to guideline, prepared with ultra-pure water

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Complete darkness

EFFECT PARAMETERS MEASURED: Immobility after 24 and 48 hours of exposure. In addition to immobility, any abnormal behaviour or appearance was reported.

RANGE-FINDING STUDY
In order to select appropriate test concentrations for use in the definitive test, a non-GLP preliminary range-finding tests was conducted to determine the approximate toxicity of the test item. Due to the test item’s low solubility, preparation of test solutions was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23) as follows: In the semi-static preliminary range-finding test the nominal test concentration of 100 mg/L was prepared and handled in ultrasonic bath for 10 minutes. The stock solution was mixed for a period of 24 hours to achieve an equilibrated concentration and then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. The test solution was renewed once during the test (on day 1). Untreated control ran parallel in the test.
- Test concentration: 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. Based on the results of the non-GLP Preliminary Range-Finding Tests, the main test was conducted as a limit test using the WAF method (according to OECD Series on Testing and Assessment No. 23) including a loading rate of 100 mg test item/L and a concurrent control group.

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 1.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Validity of the Study

Immobilisation was not observed in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was in the range of 7.47 - 8.65 mg/L (more than 3 mg/L in all cases). All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Analytical Results

The concentration of the test item was analytically determined based on total product at the start and at the end of each renewal period of the experiment.
The test item was not detected in the untreated control group (i.e. signal intensities measured for the control samples were less than 20 % of the analytical quantification limit (LOQ).
In the treated group the mean measured test item concentration was 1.62 mg/L at the start and 0.931 mg/L at the end of the first renewal period (corresponding to 57 % of the initial measured test concentration) while it was measured to be 1.55 mg/L at the start and 1.10 mg/L at the end of the second renewal period (corresponding to 71 % of the initial measured test concentration).
The exposure concentration was calculated as the geometric mean of the start and end values and determined to be 1.27 mg/L. This concentration was considered as the saturation concentration in the test medium, based on water-accommodated fractions of the test item (equivalent to 100 mg/L nominal concentration).

Biological Results and Performance of the Test

Testing units were kept in a climate chamber at a temperature of 19.9 – 20.8 °C, while temperature in the test glasses ranged between 20.1 - 20.6 °C. The pH was measured as 7.83 - 8.18 and the dissolved oxygen concentration between 7.47 - 8.65 mg/L during the study. The test was carried out in total darkness. There was no immobilisation observed in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Reported statistics and error estimates:

Statistical analysis to detect significant differences between control and treated test group on immobilization was assessed by analysis of variance (ANOVA) and appropriate statistical procedure (e.g. by Dunnett’s Test, α = 0.05).

Results of the Preliminary Range-finding Tests

Nominal concentrations (mg/L)	Preliminary Range-finding Test
	Untreated control	100 (WAF)
Number of treated animals	10	10
Number of immobilised animals (at 48h)	0	0

pH-values Measured during the Experiment

Test Group	Replicate	1^strenewal period		2^ndrenewal period
		0 h	24 h		24 h	48 h
Control	1	8.07	8.13		7.83	8.12
	2		8.16			8.12
	3		8.17			8.12
	4		8.18			8.11
Saturated test concentration (1.27 mg/L measured)	1	8.13	8.12		7.80	8.08
	2		8.13			8.06
	3		8.12			8.06
	4		8.12			8.07

Dissolved Oxygen Concentrations Measured during the Experiment

Test Group	Replicate	1^strenewal period		2^ndrenewal period
		0 h	24 h		24 h	48 h
Control	1	8.51	8.20		8.33	8.60
	2		8.20			8.42
	3		8.18			8.39
	4		8.22			8.51
Saturated test concentration (1.27 mg/L measured)	1	7.47	8.16		8.65	8.47
	2		8.27			8.38
	3		8.11			8.32
	4		8.29			8.34

 

Temperatures Measured during the Experiment

Test Group	Replicate	1^strenewal period		2^ndrenewal period
		0 h	24 h		24 h	48 h
Control	1	20.6	20.4		20.5	20.2
	2		20.4			20.2
	3		20.5			20.1
	4		20.4			20.3
Saturated test concentration (1.27 mg/L measured)	1	20.6	20.5		20.5	20.3
	2		20.5			20.1
	3		20.4			20.2
	4		20.3			20.3

Immobilization of the Test Animals

Test Group	Replicate	Number of treated animals	Number of immobilised animals
			24 h	48 h
Control	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
Saturated test concentration (1.27 mg/L measured)	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of this study, the test item Leuco Sulfur Blue 9 had no toxic effect up to aquatic saturation (i.e. limit test concentration) on Daphnia magna; the 48h LOEC is higher than the solubility level of the test item in the test medium, which corresponds to a mean measured concentration of 1.27 mg/L (based on total product). The NOEC value is equal to the mean measured test item concentration of 1.27 mg/L (based on total product).

Executive summary:

Acute toxicity of the test item Leuco Sulfur Blue 9 was assessed on Daphnia magna in a 48-hour Acute Immobilisation Test. Young Daphnia were exposed to aqueous test media containing the test item for 48 hours in a semi-static test. A limit test was performed in which the test organisms were exposed to aqueous test media containing the test item for 48 hours at the limit of its solubility in the test medium (i.e. saturation) plus a control in order to demonstrate that the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)).

The quantification of the test item Leuco Sulfur Blue 9 was performed by a previously validated analytical method by the analytical laboratory of TOXI-COOP ZRT. Samples were taken from the test concentration and the control at the start and at the end of each renewal period of the experiment and analysed by UV/VIS spectrophotometric method. The mean determined exposure concentration was 1.27 mg/L (based on total product) which was calculated as the geometric mean of the measured start and end concentrations of both renewal periods.

Healthy, young female daphnids < 24 hours of age and with known origin, history and acclimatisation conditions were used. Twenty Daphnia (divided into 4 replicates) were tested each, exposed to the saturated test concentration and in the control. Each test vessel contained approximately 80 mL test medium. Fully characterized (content, physico-chemical characteristics) ISO Medium was used as test medium.

The immobilisation of the test animals was recorded 24 and 48 hours after test start. Environmental conditions were recorded at the start and at the end of each renewal period. All measured values remained within the acceptable ranges.

There was no immobilisation in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. All validity criteria were met and therefore the study can be considered as valid.

Description of key information

In a Daphnia sp. Acute Immobilisation Test according to OECD Guideline 202 with a WAF, the test item Leuco Sulfur Blue 9 had no toxic effect up to aquatic saturation (i.e. limit test concentration); the 48h LOEC is higher than the solubility level of the test item in the test medium, which corresponds to a mean measured concentration of 1.27 mg/L (based on total product). The NOEC value is equal to the mean measured test item concentration of 1.27 mg/L (based on total product).

Additional information

Acute toxicity of the test item Leuco Sulfur Blue 9 was assessed on Daphnia magna in a 48-hour Acute Immobilisation Test. Young Daphnia were exposed to aqueous test media containing the test item for 48 hours in a semi-static test. A limit test was performed in which the test organisms were exposed to aqueous test media containing the test item for 48 hours at the limit of its solubility in the test medium (i.e. saturation) plus a control in order to demonstrate that the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)).

The quantification of the test item Leuco Sulfur Blue 9 was performed by a previously validated analytical method by the analytical laboratory of TOXI-COOP ZRT. Samples were taken from the test concentration and the control at the start and at the end of each renewal period of the experiment and analysed by UV/VIS spectrophotometric method. The mean determined exposure concentration was 1.27 mg/L (based on total product) which was calculated as the geometric mean of the measured start and end concentrations of both renewal periods.

Healthy, young female daphnids < 24 hours of age and with known origin, history and acclimatisation conditions were used. Twenty Daphnia (divided into 4 replicates) were tested each, exposed to the saturated test concentration and in the control. Each test vessel contained approximately 80 mL test medium. Fully characterized (content, physico-chemical characteristics) ISO Medium was used as test medium.

The immobilisation of the test animals was recorded 24 and 48 hours after test start. Environmental conditions were recorded at the start and at the end of each renewal period. All measured values remained within the acceptable ranges.

There was no immobilisation in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. All validity criteria were met and therefore the study can be considered as valid.