Reaction product of 4-aminophenol with 2-ethyl-6-methylbenzenamine and sodium polysulfide

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: 28 July 2020

Analytical monitoring:

yes

Details on sampling:

ANALYSIS OF THE SAMPLES
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.

Vehicle:

yes

Details on test solutions:

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: 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:

freshwater

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:

231.4 mg/L (as CaCO3)

Test temperature:

20.1 - 20.6°C

pH:

7.74 - 8.12

Dissolved oxygen:

6.79 - 8.73 mg/L

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured: 0.50 mg/L (geometric mean of the start and end values )

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Type: open
- Material, size: Glass, 100 mL, amount of test solutions: 80 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. 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 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 (see above), 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:

EC50

Effect conc.:

> 0.5 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:

NOEC

Effect conc.:

0.5 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.:

> 0.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

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 .
- Behavioural abnormalities: No
- Other biological observations: No
- Mortality of control: No
- Other adverse effects control: No
- Abnormal responses: No
- Effect concentrations exceeding solubility of substance in test medium: The WAF method was applied

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 with reference item Potassium dichromate was: 05 – 06 September 2017. 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 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 (0.50 mg/L measured)	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0

Table 2: Blue Sema 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 14, 2018)		End (February 15, 2018)
	0.604± 0.020		0.373± 0.054	62
Control	< LOD			-
		Second renewal period
100 mg/L	Start (February 15, 2018)		End (February 16, 2018)
	0.745± 0.075		0.365± 0.057	49
Control	< LOD			-

The ISO control samples showed a small absorbance. The amount of any interfering component was less than 20 % of the lowest concentration of the calibration curve. 

Validity criteria fulfilled:

yes

Conclusions:

In a 48-hour semi-static test according to EU Method C.2, OECD Guideline 202 and EPA OPPTS 850.1010 the 48-hour EC50 of the test item was determined to be > 0.5 mg/L (geometric mean measured). The test item had no toxic effect to Daphnia magna up to and including aquatic saturation of the test item.

Executive summary:

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour semi-static test according to EU Method C.2, OECD Guideline 202 and EPA OPPTS 850.1010 using the WAF method (according to OECD Series on Testing and Assessment No. 23). Twenty daphnids (divided into 4 replicates) were tested each, exposed to the saturated test concentration (nominal loading rate: 100 mg/L per renewal period) and in the blank control. Each test vessel contained approximately 80 mL test medium (ISO Medium). The quantification of the test item was performed by UV/VIS spectrophotometry. The mean determined exposure concentration was 0.50 mg/L which was calculated as the geometric mean of the measured start and end concentrations of both renewal periods. 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. Suitability of the test system was confirmed by positive control with reference item potassium dichromate (24-hour EC50 = 1.53 mg/L). In result, 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. In conclusion, the test item had no toxic effect up to and including aquatic saturation (i.e.limit test concentration) on Daphnia magna; the 48-hour EC10, EC20, EC50 results and the LOEC are higher than the solubility level of the test item in the test medium, which correspondsto the mean measured concentration of 0.50 mg/L. The NOEC value is equal to the mean measured test item concentration of 0.50 mg/L.

Description of key information

In a 48-hour semi-static test according to EU Method C.2, OECD Guideline 202 and EPA OPPTS 850.1010 the 48-hour EC50 of the test item was determined to be > 0.5 mg/L (geometric mean measured, corresponding to a nominal loading rate of 100 mg/L). The test item had no toxic effect to Daphnia magna up to and including aquatic saturation of the test item.

Additional information

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour semi-static test according to EU Method C.2, OECD Guideline 202 and EPA OPPTS 850.1010 using the WAF method (according to OECD Series on Testing and Assessment No. 23). Twenty daphnids (divided into 4 replicates) were tested each, exposed to the saturated test concentration (nominal loading rate: 100 mg/L per renewal period) and in the blank control. Each test vessel contained approximately 80 mL test medium (ISO Medium). The quantification of the test item was performed by UV/VIS spectrophotometry. The mean determined exposure concentration was 0.50 mg/L which was calculated as the geometric mean of the measured start and end concentrations of both renewal periods. 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. Suitability of the test system was confirmed by positive control with reference item potassium dichromate (24-hour EC50 = 1.53 mg/L). In result, 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. In conclusion, the test item had no toxic effect up to and including aquatic saturation (i.e.limit test concentration) on Daphnia magna; the 48-hour EC10, EC20, EC50 results and the LOEC are higher than the solubility level of the test item in the test medium, which correspondsto the mean measured concentration of 0.50 mg/L. The NOEC value is equal to the mean measured test item concentration of 0.50 mg/L.