Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and 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:

11 September - 17 December 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:

2000, September

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Date of production: 04 August 2016
Expiry date: 04 August 2021

Analytical monitoring:

yes

Details on sampling:

A supersaturated solution (100 mg/L nominal loading) was prepared by adding an amount of 0.0522 g test item to 522 mL test medium (ISO Medium) in the first renewal period and 0.0465 g test item in 465 mL test medium (ISO Medium) in the second renewal period one day before the start of each renewal period. The test solution was handled by ultrasonic bath for 10 minutes, thereafter stirred for a period of 24 hours to achieve an equilibrated concentration.

Vehicle:

yes

Details on test solutions:

As the test item is poorly soluble in deionized water as well in the test medium, for preparation of test solution the water-accommodated fraction (WAF) approach was taken, as described in OECD Guidance Document No. 23. A supersaturated solution (100 mg/L nominal loading) was prepared by adding an amount of 0.0522 g test item to 522 mL test medium (ISO Medium) in the first renewal period and 0.0465 g test item in 465 mL test medium (ISO Medium) in the second renewal period one day before the start of each renewal period. The test solution was handled by ultrasonic bath for 10 minutes, thereafter stirred for a period of 24 hours to achieve an equilibrated concentration. The test solution was then filtrated through a membrane filter (0.45 µm; Thermo Nalgene® membrane) to separate the possible non-dissolved test material. The test solution was freshly prepared in the testing laboratory just before introduction of the test animals (start of each renewal period).

Controls: Negative control (ISO medium without addition of test item ), positive control (with the reference substance potassium dichromate)

Test Water (Dilution Water)
Reconstituted water (ISO Medium, according to OECD 202) was used as dilution water in the experiment. Separate stock solutions of individual substances were first prepared in deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE ELIX 3 water purification system). The ISO Medium was prepared by adding 25 mL from each of four stock solutions to one litre deionised water proportionally.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species and strain: Daphnia magna (Straus)
Origin: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
Breeding: The Daphnia are bred in the Laboratory of TOXI-COOP ZRT.
Number of animals: Twenty animals in both groups (test concentration and untreated control), divided into 4 replicates (5 animals per replicates).
Age of animals: Less than 24 h old at the beginning of the test.
Sex: Female
Animal health: Apparently healthy animals were used in this test with a known history
Acclimatization: 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. Brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.
Food and Feeding: The Daphnia were fed with concentrated algal suspension of Raphidocelis subcapitata during the holding. Test animals were not fed during the exposure.

Test type:

semi-static

Water media type:

other: ISO Medium, according to OECD 202

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Twenty animals, divided into four replicates (glass beaker; volume app. 100 mL) of five animals each (at least 4 mL test solution/animal) were exposed to the test concentration or included as control over a period of 48 hours.

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 are considered to be immobile.

Hardness:

243.86 mg/L (as CaCO3).

Test temperature:

21.2 - 21.4°C

pH:

7.57 - 8.02

Dissolved oxygen:

6.59 - 7.40 mg/L

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured: 0.188 mg/L (geometric mean of the start and end values )

Details on test conditions:

The test vessels were kept in a climate chamber under controlled environmental conditions during the test and test solutions were not aerated. The water temperature, dissolved oxygen concentration and pH were measured in each test group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of each renewal period. Additionally, the ambient temperature was measured continuously using a min/max thermometer in the climate chamber.
Test type: Semi-static test. Based on the results obtained during analytical method validation (Study number: 805-100-3112) the test item is not stable for the duration of 48 hours in ISO Medium and not remains within 80 – 120% in comparison to that of the measured test item concentration at the start of the test throughout exposure. Therefore the test solution (as well control solution) was renewed once during the test (on day 1).
Temperature: The test temperature was in the range of 21.2 - 21.4°C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 19.3 – 22.0°C.
Oxygen Concentration: The dissolved oxygen concentration was in the range of 6.59 - 7.40 mg/L during the test.
pH: The pH of the test solution was not adjusted and did not vary by more than 1.5 units in any one test. The pH was in the range of 7.57 - 8.02 during the test.
Light-Dark Cycle: The experiment was carried out in complete darkness, as described by OECD 202.
Hardness: The measured hardness of the ISO Medium used in the study was 243.86 mg/L (as CaCO3).

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.188 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: no toxic effect up to and including aquatic saturation (i.e. limit test concentration) on Daphnia magna

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 0.188 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: no toxic effect up to and including aquatic saturation (i.e. limit test concentration) on Daphnia magna

Details on results:

Validity of the Study

Immobilisation was 0.0 % (< 10 %) in the control group and the dissolved oxygen concentration during the test in control and test vessels was in the range of 6.59 - 7.40 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 ≤ 15 % of the lowest concentration of the calibration curve).In the treated group the mean measured test item concentration was 0.210 mg/L at the start and 0.108 mg/L at the end of the first renewal period (corresponding to 51 % of the initial measured test concentration) while it was measured to be 0.335 mg/L at the start and 0.164 mg/L at the end of the second renewal period (corresponding to 49 % 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 0.188 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 19.3 – 22.0 °C, while temperature in the test glasses ranged between 21.2 – 21.4°C. The pH was measured as 7.57 - 8.02 and the dissolved oxygen concentration between 6.59 - 7.40 mg/L during the study. The test was carried out in total darkness. Immobilisation was observed neither in the control group nor in the test item treated group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Results with reference substance (positive control):

For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 392-202-3732) with reference item Potassium dichromate was: 28 February – 01 March 2018. The 24h EC50 value was determined to be 1.41 mg/L (95% conf. limits: 1.19 – 1.68 mg/L), which falls within the range of 0.6 mg/L and 2.1 mg/L as given in the guideline.

Reported statistics and error estimates:

A limit test was performed and toxic effects were not observed, therefore statistical analysis was not necessary. The NOEC and LOEC values of the test item were determined directly from the raw data.

Summary of the Biological Endpoints

Endpoints	Concentration^1
48 h-NOEC	0.188 mg/L
48 h-LOEC	> 0.188 mg/L

pH-values Measured during the Experiment

Test Group	Replicate	pH
		1^strenewal period		2^ndrenewal period
		0 h	24 h		24 h	48 h
Control	1	7.71	7.86		7.57	7.89
	2		7.99			7.94
	3		7.99			7.93
	4		7.99			7.93
Saturated test concentration (0.188 mg/L measured)	1	7.94	7.84		8.02	7.82
	2		7.93			7.86
	3		7.95			7.88
	4		7.97			7.91

Dissolved Oxygen Concentrations Measured during the Experiment

Test Group	Replicate	Dissolved Oxygen Concentration (mg/L)
		1^strenewal period		2^ndrenewal period
		0 h	24 h		24 h	48 h
Control	1	7.40	7.14		7.37	7.27
	2		7.35			7.25
	3		7.35			7.27
	4		7.34			7.29
Saturated test concentration (0.188 mg/L measured)	1	6.59	7.36		6.74	7.23
	2		7.30			7.19
	3		7.28			7.18
	4		7.27			7.17

Temperatures Measured during the Experiment

Test Group	Replicate	Temperature (°C)
		1^strenewal period		2^ndrenewal period
		0 h	24 h		24 h	48 h
Control	1	21.4	21.3		21.4	21.3
	2		21.2			21.2
	3		21.2			21.3
	4		21.3			21.3
Saturated test concentration (0.188 mg/L measured)	1	21.3	21.3		21.3	21.4
	2		21.3			21.3
	3		21.4			21.2
	4		21.4			21.2

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 (0.188 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 Brown 46 had no toxic effect up to and including 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 0.188 mg/L (based on total product). The NOEC value is equal to the mean measured test item concentration of 0.188 mg/L (based on total product). The EC50 was determined to be > 0.188 mg/L.

Executive summary:

Acute toxicity of the test item Leuco Sulphur Brown 46 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 animals 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 Sulphur Brown 46 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 0.188 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 50 mL test medium. Fully characterized (content, physico-chemical characteristics) ISO Medium was used as test medium. The immobilisation of the test animals was observed 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. Immobilisation was observed neither in the control group nor in the test item treated group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

In conclusion, the test item had no toxic effect up to and including 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 the mean measured concentration of 0.188 mg/L and a nominal loading rate of 1000 mg/L. The NOEC value is equal to the mean measured test item concentration of 0.188 mg/L and 100 mg/L in nominal. All validity criteria were met and therefore the study can be considered as valid.

Description of key information

Based on the results of an Acute Immobilisation test with Daphnia magna, the test item Leuco Sulfur Brown 46 had no toxic effect up to and including 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 0.188 mg/L (based on total product). The NOEC value is equal to the mean measured test item concentration of 0.188 mg/L (based on total product). The EC50 was determined to be > 0.188 mg/L.

Additional information

Acute toxicity of the test item Leuco Sulphur Brown 46 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 animals 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 Sulphur Brown 46 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 0.188 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 50 mL test medium.Fully characterized (content, physico-chemical characteristics) ISO Medium was used as test medium. The immobilisation of the test animals was observed 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. Immobilisation was observed neither in the control group nor in the test item treated group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

In conclusion, the test item had no toxic effect up to and including 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 the mean measured concentration of 0.188 mg/L and a nominal loading rate of 1000 mg/L. The NOEC value is equal to the mean measured test item concentration of 0.188 mg/L and 100 mg/L in nominal. All validity criteria were met and therefore the study can be considered as valid.