Tall Oil Fatty Acids, compounds with 2-(2-aminoethoxy) ethanol

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:

21 May 2015 to 18 January 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 1.6 mg/L loading rate WAF test vessel at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) for quantitative analysis.
- Samples were stored frozen prior to analysis.
- Duplicate samples at 0, 24, 72 and 96 hours were stored frozen for further analysis if necessary.
- Samples at 24 hours (fresh media), 48 hours (old and fresh media) and 72 hours (old media) were also 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.

VALIDATION OF MIXING PERIOD
- Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF (see Appendix 3, attached).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

- 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 from 07 October 2015.
- Fish were maintained in a glass fibre tank with a 'single pass' water renewal system.
- Fish were acclimatised to test conditions from 26 October 2015 to 02 November 2015.
- The lighting cycle was controlled to give 16 hours light and 8 hours darkness with 20 minute dusk and dawn periods.
- Water temperature was controlled at approximately 13 °C with a dissolved oxygen content of ≥ 9.2 mg O2/L. These parameters were recorded daily.
- The stock fish were fed commercial trout pellets, which were discontinued approximately 24 hours prior to the start of the definitive test.
- There were no mortalities in the 7 days prior to the start of the test and the fish had a mean standard length of 4.4 cm (sd = 0.4) and a mean weight of 1.13 g (sd = 0.31) at the end of the definitive test. Based on mean weight value this gave a loading rate of 0.39 g bw/L.
- The diet and diluent water were not considered 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

Post exposure observation period:

Not applicable

Hardness:

Approximately 140 mg/L as CaCO3

Test temperature:

14 - 15 °C

pH:

7.7 to 8.2 (see Table 2, attached)

Dissolved oxygen:

8.9 to 10.2 mg O2/L (see Table 2, attached)

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentration 1.6 mg/L

Details on test conditions:

DEFINITIVE TEST
- In accordance with the recommendations of REACH, the test was conducted according to the threshold approach recommended by ECHA.
- Using this approach the lowest EL50 value from either the algal growth inhibition study or acute toxicity to Daphnia magna study is set as the threshold loading rate and a "limit test" is conducted at this threshold loading rate.
- If no mortalities are observed this indicates that fish are not the most sensitive species and that the LL50 is greater then the loading threshold rate. - The EL50 value obtained from the acute toxicity to Daphnia magna study was the lower of the two applicable EL50 values and hence the test was conducted at a single loading rate of 1.6 mg/L.

EXPERIMENTAL PREPARATION
- A nominal amount of test item (35.2 mg) was added to the surface of 22 L of test water to give the 1.6 mg/L loading rate.
- After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- Stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour.
- A wide bore glass tube, covered at one end with Nescofilm, was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel.
- A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal.
- A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.6 mg/L loading rate WAF.

EXPOSURE CONDITIONS
- Glass exposure vessels (25 to 30 L) containing test media (20 L) were used for each control and test concentration.
- At the start of the test, seven fish were placed in the test preparations in each test vessel at random.
- Test vessels were then covered to reduce evaporation and maintained at 14 to 15 °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.
- Test vessels were aerated via narrow bore glass tubes.
- 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 which daily renewal of the test preparations took place to prevent build up of nitrogenous waste products.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

VALIDATION OF MIXING PERIOD
- Preliminary investigational work (see Appendix 3, attached) indicated that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours.
- Therefore, for the purpose of testing, the WAF was prepared using a stirring period of 23 hours followed by a 1-hour settlement period.

CHEMICAL ANALYSIS OF TEST LOADING RATES
- Analysis of the freshly prepared test preparation at 0 and 72 hours (see Appendix 4, attached) showed that measured concentrations of 1.33 and 1.24 mg/L were obtained respectively.
- The old or expired test item at 24 and 96 hours showed that measured concentrations of 1.15 and 0.0048 mg/L were obtained respectively.
- A low measured concentration was observed in the old test media at the 96-hour time point and analysis of the duplicate sample confirmed the result. This finding could have been due to the media being observed as a cloudy dispersion at 96 hours with the lower measured concentration resulting from test item coming out of solution at this stage. However, given that results were based on nominal concentrations, there was considered to be no impact on the outcome or integrity of the test.
- Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates 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).
- There were no mortalities in 7 fish exposed to a 1.6 mg/L loading rate WAF for a period of 96 hours.
- Inspection of mortality data gave LL50 values > 1.6 mg/L loading rate WAF at 3, 6, 24, 48, 72 and 96 hours.
- The No Observed Effect Loading Rate (NOELR) was considered to be 1.6 mg/L loading rate WAF.

SUB-LETHAL EFFECTS
- There were no sub-lethal effects of exposure observed in 7 fish exposed to a 1.6 mg/L loading rate WAF 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 ≥ 60 % of ASV (6.2 mg O2/L) in the control and test vessels.

WATER QUALITY CRITERIA
- Results of the water quality measurements are given in Table 2 (attached).
- Temperature was maintained at 14 to 15 °C throughout the test.
- There were no treatment related differences for oxygen concentration or pH.

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- Observations on the test media were carried out during the mixing and testing of the WAF.
- At the start of the mixing period the 1.6 mg/L loading rate was observed to be a clear colourless water column with test item floating at the surface.
- After 23 hours stirring and a 1-hour standing period the 1.6 mg/L loading rate was observed to be a clear colourless water column.
- Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
- In all fresh media, and in the old media at 24 and 48 hours, the 1.6 mg/L loading rate was observed to be a clear, colourless solution.
- In the 72 and 96-hour old media, the 1.6 mg/L loading rate was observed to be a very slightly or slightly cloudy dispersion.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) was investigated in accordance with OECD Guideline 203 using the threshold approach and gave a 96 -hour LL50 value of greater than 1.6 mg/L loading rate WAF. The No Observed Effect Loading rate was 1.6 mg/L loading rate WAF.

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

Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF).

In accordance with REACH, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EL50 value from either the algal growth inhibition study or acute toxicity to Daphnia magna study is set as the threshold loading rate and a "limit test" is conducted at this threshold loading rate. If no mortalities are observed this indicates that fish are not the most sensitive species and that te LL50 is greater than the threshold loading rate. The EL50 value obtained from investigation of acute toxicity to Daphnia magna was the lowest of the two EL50 values and hence the test was conducted at a single loading rate of 1.6 mg/L.

Seven fish were exposed to a Water Accommodated Fraction (WAF) of the test item at a single nominal loading rate of 1.6 mg/L for a period of 96 hours at a temperature of 14 °C to 15 °C under semi-static conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination at 96 hours.

RESULTS

Analysis of the test preparations at 0 and 72 hours (fresh media) showed that measured concentrations of 1.33 and 1.24 mg/L were obtained respectively.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of rainbow trout to the test item gave LL50 values of greater than 1.6 mg/L loading rate WAF. The No Observed Effect Loading Rate was 1.6 mg/L loading rate WAF.

CONCLUSION

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) was investigated using the threshold approach and gave a 96 -hour LL50 value of greater than 1.6 mg/L loading rate WAF. The No Observed Effect Loading rate was 1.6 mg/L loading rate WAF.

Description of key information

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) was investigated in accordance with OECD Guideline 203 using the threshold approach and gave a 96 -hour LL50 value of greater than 1.6 mg/L loading rate WAF. The No Observed Effect Loading rate was 1.6 mg/L loading rate WAF.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.6 mg/L

Additional information

No additional data.