N,N,4-trimethylpiperazine-1-ethylamine

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 04 December 2017 and 16 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

EC No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: Jeffcat TAP
- Chemical name: N,N,4-Trimethylpiperazine-1-ethylamine
- Molecular weight: 171.29 g/mol
- Batch: PFW160141
- CAS number: 104-19-8
- Purity: 99.5%
- Physical state/Appearance: Clear colourless liquid
- Expiry Date: 30 December 2018
- Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Water samples were taken from the control and the 29 mg/L test vessel at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis. The samples were stored frozen prior to analysis.
- Sample storage conditions before analysis: Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (580 mg) was dissolved into 1 liter of test water to give a 580 mg/L stock solution. The pH of this solution was then adjusted bring it within the range of the diluent as quoted in the study plan of between 6 and 9 using 0.1 M hydrochloric acid. The entirety of this stock solution was then added to the 25 – 30 liter test vessel and made up to 20 liters with test water to give the 29 mg/L test concentration.
The stock solution was inverted several times to ensure adequate mixing and homogeneity and the test solution was mixed with a flat bladed stirrer for 1 minute to ensure adequate mixing and homogeneity, however, due to a technical oversight this was not performed during the preparation of the 24 hour solution. This was considered not to have adversely affected the outcome of the study as analytical results showed nominal concentrations were achieved.
The concentration and stability of the test item in the test preparations were verified by chemical analysis of the fresh media at 0 and 72 hours and of the old media at 24 and 96 hours

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 12 September 2017.
- Length at study initiation (length definition, mean, range and SD): There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.7 cm (standard deviation = 0.1)
- Weight at study initiation (mean and range, SD): mean body weight of 1.47 g (standard deviation = 0.09) at the end of the definitive test. Based on the mean body weight value this gave a loading rate of 0.51 g body weight/liter.

ACCLIMATION
- Acclimation period: Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 27 November 2017 to 04 December 2017. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Type and amount of food during acclimation: The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

approximately 140 mg/L as CaCO3

Test temperature:

14°C

pH:

7.1 - 8.1

Dissolved oxygen:

DO content of greater than or equal to 9.9 mg O2/L

Nominal and measured concentrations:

Analysis of the test preparations at 0, 24, 72 and 96 hours showed measured test concentrations to be near nominal and so it was considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only.
Analytical results of the control samples at both 72 and 96 hours showed that low levels of test item were present. These were 0.018 and 0.020 mg/L respectively. This was considered not to have adversely affected the outcome of the study as none of the control fish died or showed any signs of stress.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass
- Type (delete if not applicable): closed , test vessels were covered to reduce evaporation and maintained at approximately 14”C in a temperature controlled room
- Material, size, headspace, fill volume: 25 – 30 liter glass exposure vessels containing 20 liters of test media
- Aeration: test vessels were aerated via narrow bore glass tubes
- Renewal rate of test solution (frequency/flow rate): no, static regime
- No. of organisms per vessel: At the start of the test 7 fish were placed in each test vessel at random, in the test preparations.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Culture medium different from test medium: The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or the 24 Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : an estimate of the LC50 values was given by inspection of the mortality data. Any mortalities and sub-lethal effects of exposure were recorded at 1, 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: only one concentration (limit test)

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0, 24, 72 and 96 hours showed measured test concentrations to be near nominal and so it was considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only.
Analytical results of the control samples at both 72 and 96 hours showed that low levels of test item were present. These were 0.018 and 0.020 mg/L respectively. This was considered not to have adversely affected the outcome of the study as none of the control fish died or showed any signs of stress.

Mortality Data
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2.
There were no mortalities in 7 fish exposed to a test concentration of 29 mg/L for a period of 96 hours.

Sub-Lethal Effects
There were no sub lethal effects of exposure observed in 7 fish exposed to a test concentration of 29 mg/L for a period of 96 hours.

alidation Criteria
The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was equal to or greater than 60% of ASV (6.1 mg O2/L at 15 °C) in the control and test vessels.

Water Quality Criteria
Temperature was maintained at approximately 14”C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Observations on Test Item Solubility
The test item preparations were observed to be clear colorless solutions throughout the test.

Sublethal observations / clinical signs:

Table 2       Cumulative Mortality Data in the Definitive Test 

Nominal Concentration (mg/L)	Cumulative Mortality (Initial Population = 7)							Mortality (%)
	1 Hour	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0	0
29	0	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and based on the nominal test concentration gave a 96 Hour LC50 of greater than 29 mg/L. The No Observed Effect Concentration was 29 mg/L.

Executive summary:

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods

In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EC₅₀value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC₅₀ is greater than the threshold concentration. The EC₅₀value obtained from the Algal Growth Inhibition study (Envigo study number XW72HY) was the lowest of these two EC₅₀values and hence the test was conducted at a single concentration of 29 mg/L.

Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 29 mg/L for a period of 96 hours at a temperature of approximately 14 °C under semi‑static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results…….

Analysis of the test preparations at 0, 24, 72 and 96 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC₅₀ values based on the nominal test concentrationsof greater than 29 mg/L. The No Observed Effect Concentration (NOEC) was 29 mg/L.

 

Description of key information

The study of Squance (2018), investigating the acute toxicity of the test substance in rainbow trout according to OECD guideline 203, was considered as the key study for endpoint coverage. An LC50 > 29 mg/L (based on nominal test concentrations) was obtained after an exposure of 96 hours in a static test in freshwater. Based on the results it can be concluded that the test substance is not harmful to fish.