2,5-cyclohexadien-1-one, 4-[[4-(phenylamino)phenyl]imino]-, 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:

31 January - 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:

Date of production: 08 July 2015
Expiry date: 08 July 2020

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Four replicate samples were analysed from the test solutions at the start and at the end of the renewal periods. Four replicate samples were analysed from the control as well.. The first series of the samples taken at the start and at the end of each renewal was sent to individual analysis.
- Analytical sampling dates: 06 February 2018 (start of experiment, start of 1st renewal period), 07 February 2018 (end of 1st renewal period , start of 2nd renewal period), 08 February 2018 (end of 2nd renewal period, end of experiment)
- Sample storage conditions before analysis: At a temperature below – 15 °C.

Vehicle:

no

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, 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 0.0703 g test item to 703 mL test medium (ISO Medium) in the first renewal period and 0.0700 g test item in 700 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 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: No

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. The test animals were not fed during the test. Immobility or mortality was observed by visually after 24 and 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 within15 seconds after gentle agitation of the test beaker were considered to be immobile.

Hardness:

The measured hardness of the ISO Medium used in the study was 249.2 mg/L (as CaCO3).

Test temperature:

The test temperature was in the range of 20.4 - 20.6°C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 20.3 – 20.9°C.

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 7.93 - 8.16 during the test.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 7.32 - 8.41 mg/L during the test.

Salinity:

Not applicable

Conductivity:

Not determined

Nominal and measured concentrations:

Nominal: 100 mg/L
Geometric mean measured: 0.45 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Type: open
- Material, size: Glass, volume app. 50 mL
- Aeration: No
- 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.

The preliminary range-finding tests were not performed in compliance with the GLP-Regulations and will be excluded from the Statement of Compliance (Statement of the Study Director) in the Final Report, but the raw data of these tests will be archived under the study code of the present study.

- Test concentrations: 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 (see above), the main test is 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:

EC50

Effect conc.:

> 0.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 0.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 0.45 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:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL10

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.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

VALIDITY OF THE TEST
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 7.32 - 8.41 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.

Results with reference substance (positive control):

The 24h EC50 value was determined to be 1.53 mg/L (95% conf. limits: 1.30 – 1.78 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.

Table 1: Immobilisation of 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 (0.45 mg/L measured)	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0

Table 2: Leuco Sulfur Blue 13 Concentrations Measured during the Study

Sample Code	Mean of the measured concentrations (mg/L) with the 95% confidence intervals (n=4)		Percentage of the start concentration
100 mg/L	First renewal period
	Start (February 06, 2018)	End (February 07, 2018)
	0.732± 0.090	0.272± 0.018		37
Control	< LOD			-
	Second renewal period
100 mg/L	Start (February 07, 2018)	End (February 08, 2018)
	0.749± 0.082	0.283± 0.106		38
Control	< LOD			-

Validity criteria fulfilled:

yes

Conclusions:

No toxic effects of the test item were recorded up to the solubility limit of the test item in the test medium, which corresponds to 0.45 mg/L (geometric mean measured) and a nominal loading rate of 100 mg/L. Therefore, the 48-hour EC50 was determined to be > 0.45 mg/L (geometric mean measured).

Executive summary:

The acute toxicity of the test item to Daphnia magna was determined in a GLP-compliant 48‑hour semi-static test according to OECD Guideline 202, EU Method C.2 and EPA OPPTS 850.1010. Test animals were exposed to aqueous solutions containing the test item at the limit of its solubility in the test medium (i.e. saturation; equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)). The test item concentration was analyzed by UV/VIS spectrophotometry. The geometric mean test item concentration was determdined to be 0.45 mg/L. Healthy, young female daphnids < 24 hours of age and with known origin, history and acclimatisation conditions were used. Twenty daphnids (divided into 4 replicates) were exposed to the saturated test concentration and to the blank control (containing ISO Medium only). 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. In result, there was no immobilisation in twenty daphnids exposed to either test item treated or control group. Furthermore, no abnormal behaviour or appearance of test animals was detected. In conclusion, the test item had no toxic effect up to and including the aquatic saturation (i.e.limit test concentration) on Daphnia magna. Hence, the 48h EC10, EC20 and EC50 results and the LOEC are higher than the solubility level of the test item in the test medium, which corresponds to the mean measured concentration of 0.45 mg/L and a nominal loading rate of 100 mg/L. The NOEC value is equal to the geometric mean measured test item concentration of 0.45 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

No toxic effects of the test item were recorded up to the solubility limit of the test item in the test medium, which corresponds to 0.45 mg/L (geometric mean measured) and a nominal loading rate of 100 mg/L. Therefore, the 48-hour EC50 was determined to be > 0.45 mg/L (geometric mean measured).

Additional information

The acute toxicity of the test item to Daphnia magna was determined in a GLP-compliant 48‑hour semi-static test according to OECD Guideline 202, EU Method C.2 and EPA OPPTS 850.1010. Test animals were exposed to aqueous solutions containing the test item at the limit of its solubility in the test medium (i.e. saturation; equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)). The test item concentration was analyzed by UV/VIS spectrophotometry. The geometric mean test item concentration was determdined to be 0.45 mg/L. Healthy, young female daphnids < 24 hours of age and with known origin, history and acclimatisation conditions were used. Twenty daphnids (divided into 4 replicates) were exposed to the saturated test concentration and to the blank control (containing ISO Medium only). 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. In result, there was no immobilisation in twenty daphnids exposed to either test item treated or control group. Furthermore, no abnormal behaviour or appearance of test animals was detected. In conclusion, the test item had no toxic effect up to and including the aquatic saturation (i.e.limit test concentration) on Daphnia magna. Hence, the 48h EC10, EC20 and EC50 results and the LOEC are higher than the solubility level of the test item in the test medium, which corresponds to the mean measured concentration of 0.45 mg/L and a nominal loading rate of 100 mg/L. The NOEC value is equal to the geometric mean measured test item concentration of 0.45 mg/L and 100 mg/L in nominal. All validity criteria were met and therefore the study can be considered as valid.