Fatty acids, tallow, compds. with triethanolamine

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:

03 June 2016 to 18 August 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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control samples and test sample at nominal concentration of 100 % saturated solution. In each case, duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required. Concentrations of the test material were determined by initially diluting the liquid sample 1:1 with tetrahydrofuran, then diluting further with diluent as required to bring the response within the plotted range. Samples are analysed by injection onto a liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) system.
- Sampling method: Samples (ca. 20 mL) were taken from the control and test concentration at 0 and 72 hours (fresh media), and 24 and 96 hours (old media) for analysis. In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Initial solubility work carried out indicated that the test substance solubility was poor. Therefore, It was considered justifiable to carry out a media trial using a saturated solution preparation method, analytical samples were also taken for analysis to confirm solubility.
The variability and inconsistency of achieved concentration observed during the range-finder and definitive phases of the study were considered to be due to the poor solubility of the compound, the UVCB (Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Materials) nature of the compound, in addition, it is considered that filtration of the saturated solutions may have also impacted on the achieved concentrations as there was potential for micro-globules or micelles to pass through the filters.
At 24, 72 and 96 hours results analytical concentrations were below the LOQ and therefore, could not be reported, therefore, concentration below the LOQ were given a 1’2 LOQ value for the for the calculation of geometric mean measured concentrations (guidance from OECD 27). However, a geometric mean cannot be calculated with two concentrations that are the same at each time point, therefore, the 96 hour result was considered to be 0.049 mg/L to give the smallest decline in concentration possible purely for the calculation of the geometric mean measured concentrations.
- Method: At the start of the test, ca. 1500 mg of the test substance was added to 15 L of treated mains water to give 100 mg/L top concentration. This top concentration was then stirred for ca. 24 hours. After stirring the top concentration was then filtered through a 0.45µm filter to give a 100 % saturated solution. The remaining concentrations were achieved by serially diluting from the filtered top concentration.
- Controls: The control was prepared by adding treated mains water only to the control vessel.
- Chemical name of vehicle: Not applicable, the test material was added to water.
- Evidence of undissolved material: The control media was observed to be colourless solutions throughout the duration of the test.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Length at study completion: The total length and wet weight of ten randomly selected fish from the stock fish were determined periodically to ensure fish were of a suitable size and weight for testing and to ensure the correct testing volumes were used so as not to exceed the recommended loading rate. A sub-sample of ten fish taken at random from all surviving fish after 96 hours gave a mean total length of 5.2 cm and the length ranged between 4.7 and 5.8 cm.
- Weight at study completion: Sub-sample of ten fish taken at random from all surviving fish after 96 hours gave a mean total weight of 1.30 g and the weight ranged from 0.93 to 1.73 g.
- Maintenance of the fish: The fish were held in a temperature controlled room under artificial light, with a 16 hour light: 8-hour dark photo-period, in holding tanks at a density appropriate to their size, under continuous water renewal (flow-through) conditions.

ACCLIMATION
- Acclimation period: Fish were selected, at random, from the holding tank and allocated to the test vessels at the start of each phase of testing. The fish were acclimatised to the test conditions for at least 12 days prior to testing.
- Acclimation conditions: Same as test.
- Type and amount of food during acclimation: The fish were fed with a proprietary fish food, which was added to the holding tank in quantities dictated by the size of the fish.
- Feeding frequency during acclimation: On a weekly basis during the holding period, a sub sample of 10 fish was weighed and a mean wet weight calculated. The wet weight was used to determine the quantity of food required for the number of fish remaining in the holding tank. The amount of food required per holding tank was weighed and stored in labelled feed containers place in the fish holding room. The feed was distributed to the holding tank over two feeding intervals during the day. Uneaten food and debris was siphoned or cleaned from the tanks as required.
- Health during acclimation: Fish mortalities in the holding tank were recorded as they occurred. Use of the fish for testing was considered acceptable if the cumulative mortality in the batch was <5 % during the 7 day period preceding the proposed test start date. If the mortality exceeded 5 % mortality in this same period, or disease was apparent, the batch of fish was not to be used for testing and acclimation would be increased for at least another 7 days before mortality rate re-assessment for testing. The mortality rate of the stock batch of fish was <5 % in the seven days prior to the definitive test.

FEEDING DURING TEST
- Feeding: The fish were not fed for at least 24 hours before the start of the definitive test and were not fed throughout the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

62 – 67 mg/L

Test temperature:

13.3 – 16.5 °C

pH:

7.17 – 7.20

Dissolved oxygen:

80 – 94 %

Nominal and measured concentrations:

Definitive test:
Nominal concentrations of 100 % saturated solution. (Loading rate 100 mg/L)
Geometric mean measured concentrations: 0.0617 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 20 L constructed glass aquaria
- Type: closed. The test vessel was fitted with a plastic lid.
- Material, size, headspace, fill volume: 15 litres of media.
- No. of organisms per vessel: Seven fish per vessel. The protocol stated that in the event of a limit test 10 fish will be added the control and test concentration. However, the decision to use 7 fish per tank was made to reduce numbers of fish used. This was not considered to impact on study integrity as the guideline requirements were still met.
- No. of vessels per concentration (replicates): One vessel per concentration.
- No. of vessels per control (replicates): One vessel per concentration.
- Biomass loading rate: 0.61 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for the holding of fish stocks and for the toxicity tests was laboratory mains supply. The dilution water (referred to hereafter as treated mains water) used for conducting the tests was treated mains water that had been passed through activated carbon filters.
- Chlorine: = 0.1 mg/L
- Alkalinity: 22.0 mg/L HCO3
- Intervals of water quality measurement: At the start of the test and at approximately 24-hour intervals during the test, the media pH, concentration of dissolved oxygen (% air saturation value) and the individual test media temperatures were recorded in the freshly prepared and old test media.
The total hardness (mg/L as CaCO3) and residual chlorine (mg/L) of the treated mains water used to prepare the test media were measured at the start of each media renewal time point.

OTHER TEST CONDITIONS
- Adjustment of pH: No pH adjustment
- Photoperiod: The test was conducted in a temperature-controlled laboratory (15 ± 2 ºC) with a 16-hour light: 8-hours dark lighting cycle with ca 30-minute dawn/dusk phases.

EFFECT PARAMETERS MEASURED:
All fish in each test vessel were observed at ca 3.5 hours after addition and then at 24-hour intervals (24, 48, 72 and 96 hours) throughout the duration of the test. The number of dead fish and those fish exhibiting toxic symptoms or modified behaviour were recorded, if applicable. Any fish exhibiting toxic symptoms at or above the substantial severity limit were humanely killed, in accordance with Schedule 1 of the UK Home Office Scientific Procedures Act (ASPA), 1986, and classed as mortalities for the following observational time point.


TEST CONCENTRATIONS
- Range finding study
At the start of the test, ca 500 mg of the test substance was added to 5 L of treated mains water to give 100 mg/L top concentration. This top concentration was then stirred for ca 24 hours. After stirring the top concentration was then filtered through a 0.45µm filter to give a 100 % saturated solution. The remaining concentrations were achieved by serially diluting from the filtered top concentration.
Single test vessels were prepared, for the control and at each test concentration, each containing three fish.
- Test concentrations: Nominal concentrations of 0.1, 1.0, 10 and 100 % saturated solution under static test conditions.
- Results used to determine the conditions for the definitive study: Yes. At 72 hours, one fish was observed being unable to swim properly and laying on its side and back at the bottom of the tank, for welfare reasons, the fish was removed and humanely killed, this was not thought to be a toxic effect. No other mortalities were observed during the range-finding test.
For the duration of the range-finding test all other fish in control and test vessels were observed as exhibiting no toxic symptoms.
Based on nominal concentrations, the results of the range-finding test suggested that the 96-hour LC50 value would be greater than 100 % saturated solution (nominal concentration).

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 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.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.062 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Details on results:

For the duration of the test, no fish from the control or top concentration test vessels showed signs of toxic symptoms.

- Observations on body length and weight:
The mean total length for all surviving fish was 5.2 cm; with minimum and maximum of 4.7 - 5.8 cm.
The mean wet weight for all surviving fish was 1.30 g, with minimum and maximum of 0.9317 - 1.7251 g.

Reported statistics and error estimates:

Statistical analysis was not carried out as no toxic effects were observed during the test. Therefore, results were derived empirically.

Sublethal observations / clinical signs:

Test Media Descriptions

The control and test media were observed to be colourless solutions throughout the duration of the test.

Validity Criteria

The validity criteria for solvent control mortality (less than 10 %) and dissolved oxygen (>60 % ASV) were both satisfied. The test was therefore considered to be valid.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 96-hour LL50 value was estimated to be > 100 mg/L and the corresponding NOEC value was 100 mg/L based on the nominal loading rate.

Executive summary:

The acute toxicity of the test material to Oncorhynchus Mykiss (Rainbow Trout), was assessed in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 203.

A study was conducted to determine the 96-hour acute toxicity of the test substance to Oncorhynchus mykiss. The study was conducted using a semi-static test design with daily renewal of test media during the test. Based on the results of a range-finding test, for which only the key findings have been reported, the definitive limit test was conducted at a nominal test concentration of 100 % saturated solution (100 mg/L). A control group was also included. Analysis of the test media samples was conducted on fresh media at 0 and 72 hours and on corresponding old media at 24 and 96 hours.

Chemistry analysis showed a decline in measured concentrations over the 24 hour renewal period at the start of the test. The results also showed inconsistent achieved concentrations between the different media renewal time points, this is thought to have been caused by several factors including, the insolubility of the compound, the UVCB (Substances of unknown or variable composition, complex reaction products or biological materials) nature of the compound, and the filtration of media due to potential of micro-globule or micelles passing through filters. As measured concentrations were not within the range of 80-120 % of nominal, the results have been reported in terms of geometric mean measured concentration, which was calculated to be 0.0617 mg/L. As results at 24 and 72 hours were below the LOQ value they were given a ½ LOQ value so that a TWM could be calculated. The 96 hour result was also below the LOQ, however, this could not be given a ½ LOQ value as this would not be possible to calculate, therefore, a value of 0.049 mg/L was given to calculate the TWM.

Under the conditions of the study, the 96-hour LL50 value was estimated to be > 100 mg/L and the corresponding NOEC value was 100 mg/L based on nominal loading rate. The LOEC value was not calculated as no effect was observed during the test.

Description of key information

Under the conditions of the study, the 96-hour LL50 value was estimated to be > 100 mg/L and the corresponding NOEC value was 100 mg/L based on nominal loading rate.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

The acute toxicity of the test material to Oncorhynchus Mykiss (Rainbow Trout), was assessed in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 203. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

A study was conducted to determine the 96-hour acute toxicity of the test substance to Oncorhynchus mykiss. The study was conducted using a semi-static test design with daily renewal of test media during the test. Based on the results of a range-finding test, for which only the key findings have been reported, the definitive limit test was conducted at a nominal test concentration of 100 % saturated solution. A control group was also included. Analysis of the test media samples was conducted on fresh media at 0 and 72 hours and on corresponding old media at 24 and 96 hours.

Chemistry analysis showed a decline in measured concentrations over the 24 hour renewal period at the start of the test. The results also showed inconsistent achieved concentrations between the different media renewal time points, this is thought to have been caused by several factors including, the insolubility of the compound, the UVCB (Substances of unknown or variable composition, complex reaction products or biological materials) nature of the compound, and the filtration of media due to potential of micro-globule or micelles passing through filters. As measured concentrations were not within the range of 80-120 % of nominal, the results have been reported in terms of geometric mean measured concentration, which was calculated to be 0.0617 mg/L. As results at 24 and 72 hours were below the LOQ value they were given a ½ LOQ value so that a TWM could be calculated. The 96 hour result was also below the LOQ, however, this could not be given a ½ LOQ value as this would not be possible to calculate, therefore, a value of 0.049 mg/L was given to calculate the TWM.

Under the conditions of the study, the 96-hour LL50 value was estimated to be > 100 mg/L and the corresponding NOEC value was 100 mg/L based on the nominal loading rate. The LOEC value was not calculated as no effect was observed during the test.