Reaction mass of 2-hydroxy-N-(2-(2-hydroxyethoxy)ethyl)-N-(2-hydroxyethyl)-N-methylethan-1-amininium hydroxide and 2-hydroxy-N,N-bis(2-hydroxyethyl)-N-methylethanaminium hydroxide

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:

2017-10-16 to 2017-10-24

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
- Source and lot/batch No.of test material: D26-3-3-1
- Expiration date of the lot/batch: 2018-12-01
- Purity test date: no data
- Purity: 47.4 wt%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data

Analytical monitoring:

yes

Details on sampling:

- Sampling method:
* Definitive Test: Water samples were taken from the control and the 100 mg/L test vessel at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative 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.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (2000 mg) was dissolved into 2 liters of dechlorinated tap water and inverted several times to ensure adequate mixing and homogeneity. A pH adjustment was required to bring it down to within the guideline criteria of between pH 6 and 9 using 1 molar hydrochloric acid. This 1000 mg/L stock solution was then added to the test vessel and made up to 20 litres. The preparation was then stirred with a flat blade mixer for approximately 1 minute.
The concentration and stability of the test item in the test preparation was verified by chemical analysis at 0, 24, 72 and 96 hours
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All solutions were clear and colourless.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Length at study initiation (length definition, mean, range and SD): mean: 4.5 cm (standard deviation = 0.3)
- Weight at study initiation (mean and range, SD): mean: 0.80 g (standard deviation = 0.25)
- Feeding during test: no

ACCLIMATION
- Acclimation period: at least 7 days after delivery
- Acclimation conditions (same as test or not): yes
- Type and amount of food: pelleted fish food
- Feeding frequency: daily
- Health during acclimation (any mortality observed): mortality less than 5% seven days before 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:

14-15 °C

pH:

7.2-8.1

Dissolved oxygen:

9.2-10.1

Salinity:

not relevant

Conductivity:

not indicated

Nominal and measured concentrations:

Final test:
Nominal concentrations: 100 mg/L
Measured concentrations (mg/L) at t=0h: Measured concentrations (mg/L) at t=24h: Measured concentrations (mg/L) at t=72h: Measured concentrations (mg/L) at t=96h:

Details on test conditions:

TEST SYSTEM
- Test vessel: exposure vessels
- Material, size, headspace, fill volume: 25-30 L, all-glass filled with 20L test solution
- Aeration: not during the test
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): not reported
- No. of vessels per control (replicates): not reported
- Biomass loading rate: 0.28 g fish/L

OTHER TEST CONDITIONS
- Photoperiod: 16h light daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered to be dead when no reaction was observed after touching the caudal peduncle and visible breathing movements were absent.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: none (limit test)
- Range finding study: no
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Mortality of control: none
- Other adverse effects control: no
- Abnormal responses: none

Reported statistics and error estimates:

An estimate of the LC50 values was given by inspection of the mortality data.

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
100	0	0	0	0	0	0	0	0

 

Validity criteria fulfilled:

yes

Remarks:

considered to be valid - 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.

Conclusions:

The acute toxicity of the test substance to Rainbow trout was determined in a 96 hour semi-static limit test according to the OECD guideline 203. Under the conditions of the present study, the 96-h LC50 exceeded the nominal concentration of 100 mg/L. The results of the test can be considered reliable without restriction.

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 EC50 value 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. Therefore, as the EC50 values obtained for both the Algal Growth Inhibition study and the Acute Toxicity to Daphnia magna study (Envigo Study Numbers XR96NX and DS29KP respectively) were greater than 100 mg/L, the test was conducted at a single concentration of 100 mg/L to ensure that toxicity was not observed at this concentration. Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 100 mg/L for a period of 96 hours at a temperature of 14 ºC to 15 °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 of greater than 100 mg/L. The No Observed Effect Concentration was 100 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. In accordance with the recommendations of REACH, the test was conducted according to the threshold approach recommended by ECHA.  Using this approach the lowest EC50 value 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. Therefore, as the EC50 values obtained for both the Algal Growth Inhibition study and the Acute Toxicity to Daphnia magna study (Envigo Study Numbers XR96NX and DS29KP respectively) were greater than 100 mg/L, the test was conducted at a single concentration of 100 mg/L to ensure that toxicity was not observed at this concentration. Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 100 mg/L for a period of 96 hours at a temperature of 14 ºC to 15 °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.

An LC50 > 100 mg/L (based on nominal test concentrations) was obtained after an exposure of 96 hours in a semi-static test in freshwater. Based on the results it can be considered that the test substance is not harmful to fish.