6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)tris-hexanoic acid, compd. with 1-amino-2-propanol

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:

22 November 2016 to 02 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Water samples were taken from the control and the 100 mg a.i./L test vessel at 0 and 72 hours (fresh media). Samples were also taken at 24 and 96 hours (old media).
- Duplicate samples were taken and stored frozen for further analysis if necessary.
- Further samples at 24 hours (fresh media) 48 hours (fresh and old media) and 72 hours (old media), were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

TEST WATER
- The test water used for the definitive test was the same as that used to maintain the stock fish.
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. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Annex 2 (attached).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST SYSTEM
- The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 10 November 2016. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 15 November 2016 to 22 November 2016. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- The water temperature was controlled at 14 °C with a dissolved oxygen content of greater than or equal to 9.5 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.8 cm (sd = 0.20) and a mean weight of 0.93 g (sd = 0.15) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.33 g bodyweight/L(static volume).
- The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total hardness approximately 140 mg/L as CaCO3

Test temperature:

14 °C

pH:

7.8 to 8.2 (see Table 3, attached)

Dissolved oxygen:

9.6 to 10.9 mg O2/L (see Table 3, attached)

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentration of 100 mg a.i./L

Details on test conditions:

DEFINITIVE TEST
- At the request of the Sponsor, for the purposes of the definitive test, all test concentrations were corrected for a test item water content of 41%.
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 were greater than 100 mg a.i./L, the test was conducted at a single concentration of 100 mg a.i./L to ensure that toxicity was not observed at this concentration.

EXPERIMENTAL PREPARATION
- A nominal amount of test item (4237 mg) was dissolved in test water and the volume adjusted to 1 litre to give the 2500 mg a.i./L stock solution. An aliquot (800 mL) of the stock solution was diluted to 20 litres of test water to give the 100 mg a.i./L test concentration.
- The stock solution was inverted several times and the test concentration was stirred for approximately 1 minute with a flat bladed stirrer to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test samples were verified by chemical analysis at 0, 24, 72 and 96 hours (see Annex 3, attached).

EXPOSURE CONDITIONS
- Glass exposure vessels of 25 to 30 L, containing 20 L of test media were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at 14 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20-minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.
The control group was maintained under identical conditions but not exposed to the test item.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build-up of nitrogenous waste products.
TEST ORGANISM OBSERVATIONS
- 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.

WATER QUALITY CRITERIA
- 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 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.

DATA VALIDATION
- The results of the test are considered valid if the following criteria are met:
(i) In the control, not more than one of the fish should die or show signs of stress during the 96 hours.
(ii) The dissolved oxygen concentration at the end of the test should be ≥ 60 % of Air Saturation Values (ASV) in the control and test vessels.

MAJOR COMPUTERISED SYSTEMS
- Building management: Delta control system

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

VERIFICATION OF TEST CONCENTRATIONS
- Analysis of the test preparations at 0, 24, 72 and 96 hours (see Annex 3, attached) 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.

MORTALITY DATA
- Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 1 (attached).
- Exposure of Rainbow Trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal test concentrations of greater than 100 mg a.i./L. The No Observed Effect Concentration was 100 mg a.i./L.

SUB–LETHAL EFFECTS
- There were no sub-lethal effects of exposure observed in seven fish exposed to a test concentration of 100 mg a.i./L for a period of 96 hours.

VALIDATION 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) in the control and test vessels.
WATER QUALITY CRITERIA
- The results of the water quality measurements are given in Table 3 (attached).
- Temperature was maintained at 14 °C throughout the test.
- There were no treatment related differences for oxygen concentration or pH.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- At the start and throughout the test the control and 100 mg a.i/L test concentration were observed to be clear colourless solutions.

Reported statistics and error estimates:

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

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Rainbow Trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal test concentrations of greater than 100 mg a.i./L. The No Observed Effect Concentration was 100 mg a.i./L.

Executive summary:

GUIDELINE

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 were greater than 100 mg a.i./L, the test was conducted at a single concentration of 100 mg a.i./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 a.i./L for a period of 96 hours at a temperature of 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.

 

CONCLUSION

Exposure of Rainbow Trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal test concentrations of greater than 100 mg a.i./L. The No Observed Effect Concentration was 100 mg a.i./L.

Description of key information

Exposure of Rainbow Trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal test concentrations of greater than 100 mg a.i./L. The No Observed Effect Concentration was 100 mg a.i./L OECD 203 and EU Method C.1).

Additional information

GUIDELINE

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 were greater than 100 mg a.i./L, the test was conducted at a single concentration of 100 mg a.i./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 a.i./L for a period of 96 hours at a temperature of 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.

 

CONCLUSION

Exposure of Rainbow Trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal test concentrations of greater than 100 mg a.i./L. The No Observed Effect Concentration was 100 mg a.i./L.