Fatty acids, tallow, compds. with triethanolamine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 December 2015 to 30 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal test concentration of 100 % saturated solution
- Sampling method: Concentration of test material in Treated Mains Water was determined by dilution followed by MS/MS Detection TOF system.
- Sample analysis: At the start of the test (0 hours), ca. 20 mL samples of freshly prepared test media were taken from the control and 100 % saturated solution test media preparation flasks for chemical analysis.
At 24 hours, ca. 20 mL samples were also taken for chemical analysis from the preparation flasks (new media), and old test media at control and 100 % saturated solution were sampled from pooled test vessels.
At 48 hours, ca. 20 mL samples were also taken for chemical analysis from the pooled old test media at control and 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.

Vehicle:

no

Details on test solutions:

PRELIMINARY SOLUBILITY TRIALS AND PREPARATION OF TEST SOLUTION
- Preliminary Solubility Trial and Media Preparation Work
Initial visual solubility work was conducted at 100 mg/L in Elendt medium, however, it was observed as not in solution after several attempts with different addition techniques including sonication and stirring (24 and 48-hour stirs). Saturated solution and WAF (water accommodated fraction) preparation methods were also investigated, however, WAF were not physically suitable and analytical results showed much variation between saturated solution samples. Therefore, it was considered justifiable to change the medium used in the study from Elendt to ASTM medium as there were concerns the media was causing issues with the analytical method. The use of acetone and Tetrahydrofuran (THF) were also investigated with little success. Therefore, a media trial was conducted using a column filled with glass beads soaked with the test substance. The column was on continuous circulation to see if a saturated solution could be achieved, however, this was unsuccessful and analytical chemistry results showed no test substance had dissolved into the medium. Another trial was also conducted using saturated solutions, but rather than filtering to remove particulate matter centrifugation was used. However, centrifugation did not succeed in removing particulate from the suspension of globules. Therefore, based on the nature of the compound, it was considered justifiable to carry out the test using a saturated solution preparation method; a media trial with analytical samples was conducted to assess water solubility.

- Test Substance Properties and Related Issues: The large variability and inconsistency of achieved concentration observed at several stages of the study, from media trials through to the range-finders/experiments and finally the definitive test of the study were considered to be due to the poor solubility of the compound, the UVCB nature of the compound, in addition, it is considered that filtration of the saturated solutions may have also impacted on the achieved concentrations.

- Method: At the start of the test, the 100 % saturated solution top concentration was prepared by weighing ca. 50 mg of test substance and adding to 500 mL of treated mains water. This was stirred for ca. 24 hours. After stirring, the test concentration was then filtered through a 0.45 µm filter. A control treatment was prepared by adding treated mains water only to the control vessels.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus
- Age: All juveniles used to initiate a test were less than 24 hours old.
- Feeding during test: The daphnia were not fed during the test.

ACCLIMATION
- Acclimation period: Juvenile daphnia, less than 24 h old, were taken from healthy parental laboratory cultures. The Daphnia magna are cultured in 600 mL glass beakers containing 500 mL of Treated Mains Water. Each vessel and its contents are referred to as a 'culture'. New cultures are initiated with juvenile Daphnia magna (less than 24 hours old), at a density of approximately 15 daphnids per litre.
- Type and amount of food: The cultures were fed a concentrated suspension of Chlorella vulgaris prepared in accordance with standard operating procedures. In addition to the algal feed the cultures are fed a vitamin solution (contained 750 mg of Thiamine hydrochloride, 10 mg of Cyanocobalamine (B12) and 7.5 mg of biotine per Litre of water) and a 47.8 mg/L sodium selenate supplement is added at a rate of 50 µL/500 mL of TMW.
- Feeding frequency: The cultures were fed daily.
- Acclimation conditions (same as test or not): Daphnia were cultured in hardened treated mains water but were treated with the test material in un-hardened water. To ensure that the daphnia would not be stressed by moving from hardened treated mains water (in cultures) to the un-hardened water, a trial was conducted where by four control vessels (treated mains water only) were set up and 5 daphnia (<24 hours old) were added to each of the vessels and observed over a 48-hour test period.
The water in each culture was renewed at least once a week. Juveniles were removed when present in cultures using a sieve. Cultures were maintained up to a maximum of 4 to 5 weeks. Juveniles for use in acute toxicity tests were collected from the second brood onwards. Approximately 24 hours before a test was set up, juveniles present in the cultures were removed and discarded. Over the next 24 hours, juveniles for use in the test were removed from the culture using a wide bore pipette and transferred to fresh culture medium. The juveniles were then left for at least 1 hour before selecting actively swimming individuals for use.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

Un-hardened water.

Test temperature:

18.5 – 20.4

pH:

6.94 - 7.56

Dissolved oxygen:

7.97 - 9.76

Conductivity:

187.8 µs/cm

Nominal and measured concentrations:

Definitive test:
- Nominal test concentration of 100 % saturated solution. (Loading rate 100 mg/L)
- Measured concentration of 1.25 mg/L (freshly prepared media at 0 hours) and 0.158 mg/L (new media analysed at 24 hours).
Due to the reduction in concentration observed over the 24-hour renewal periods it was considered justifiable to base concentration on time weighted mean measured concentrations; this was calculated to be 0.233 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers with clear petri dish lids.
- Material, size, headspace, fill volume: Approximately 50 mL.
- Renewal rate of test solution (frequency/flow rate): Test medium was prepared at 0 and 24 hours.
- No. of organisms per vessel: 5 daphnia (<24 hours old) were added to each vessel with a wide bore glass pipette to avoid damaging the animals during transfer.
- No. of vessels per concentration: Four replicate test vessels
- No. of vessels per control: Four replicate test vessels

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for the toxicity test was laboratory mains supply. The water was pumped to the laboratory through an activated carbon filter.
- Alkalinity: 29.7 mg/L HCO3
- Conductivity: 187.8 µs/cm
- Intervals of water quality measurement: The pH, dissolved oxygen concentration (% air saturation value (ASV) and mg/L) and temperatures were determined in freshly prepared test media at the start of the test and at 24 hours, and in the old media at 24 and 48 hours. Continuous temperatures were measured using a digital (min/max) thermometer in an additional vessel maintained in the study area.
At 0 and 24 hours water quality measurements were performed on new media sampled directly from preparation flasks. At 24 and 48 hours test water quality measurements were conducted using pooled replicate samples of old test media.

OTHER TEST CONDITIONS
- Adjustment of pH: No pH adjustment.
- Photoperiod: Fluorescent lighting on a 16-hour light:8-hour dark photoperiod.

EFFECT PARAMETERS MEASURED: After 24 and 48 hours, the Daphnia magna in each test vessel were observed for evidence of immobility.
The observations differentiated between mobile and immobile daphnids. An individual was considered immobile if, when the contents of the test vessel were briefly agitated, it did not swim during a 15-second period of observation. In addition, Daphnia magna submerged in the body of the test media and those that were held at the surface of the test media were also recorded.

RANGE-FINDING STUDY
- Test concentrations: The range-finding test was conducted at nominal test substance concentrations of 0.10, 1.0, 10 and 100 % saturated solution under static test conditions. A control group was also included. Due to the poor solubility of the test substance, it was considered justifiable to run the range-finder test as a limit test; four replicate test vessels were prepared for the control and 100 % saturated solution test concentration and two replicate test vessels at all other test concentrations. Based on nominal concentrations, the results of the range-finding test suggested that the 48-hour EC50 value would be greater than 100 % saturated solution.
- Results used to determine the conditions for the definitive study: Yes. Due to poor analytical recoveries in the range-finding test, which were not within the acceptance criteria of 80-120 %, it was considered justifiable to run a definitive limit test, as no biological effects were observed. The definitive limit test was conducted with a test concentration of 100 % saturated solution, a control group was also included. The test was conducted under semi-static test conditions with media preparation at 0 and 24 hours as the range-finding study suggested that the test material was unstable in the static test system.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.233 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

- Other biological observations:
Daphnia were observed at 24 and 48 hours of exposure during this study and the primary endpoint indicating adverse effects was immobility. No effects were observed in the water control or 100 % test material saturated test solutions during this study.
After 48 hours, the concentration at which no significant (=10 %) dose related immobility occurred was 100 mg/L. The NOEC was therefore 100 mg/L. The 48-hour EL50 value was determined to be >100 mg/L.

Chemical Analysis Results

The limit of quantification (LOQ) for the test material in treated mains water using this method was 0.1 mg/L.

Analysis of the test media samples was conducted at 0 and 24 hours (fresh media) and 24 and 48 hours (old media).

For the 100 % saturated solution, analysis of the freshly prepared test media at 0 hours showed achieved concentrations of 1.25 mg/L, freshly prepared test media at 24 hours showed measured concentrations of 0.158 mg/L, old test media at 24 hours showed measured concentrations <LOQ (0.05 mg/L) and the old test media at 48 hours showed measured concentrations <LOQ (0.05 mg/L).

Due to the reduction in concentration observed over the 24-hour renewal periods it was considered justifiable to base concentration on time weighted mean measured concentrations; this was calculated to be 0.233 mg/L.

Concentrations over the 24 hour renewal periods dropped below the LOQ, therefore, a ½ LOQ value was given to these results to allow for a time weighted mean calculation.

Test Media Description

The test preparations were observed to be colourless solutions throughout the duration of the test.

Validity Criteria

The validity criteria for control immobility (=10 %) and dissolved oxygen (=3 mg/L) were both satisfied. The test is therefore considered valid.

Calculation of Results

Time-weighted mean measured concentrations were calculated as follows;

 

TWM = Total area / Total days

 

Where;

TWM = Time-weighted mean

Area = Area under the exponential curve for each renewal period

 

Area is calculated as follows;

 

Area = ((C0 – C1) / (1n (C0) – 1n (C1)) x days

 

Where;

C0 = measured concentration at start of renewal period

C1 = measured concentration at end of renewal period

Days = number of days in renewal period

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 48-hour EL50 was determined to be >100 mg/L and the corresponding NOEC was determined to be 100 mg/L.

Executive summary:

The toxicity of the test material to the freshwater invertebrate, Daphnia magna, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 202.

Based on the results of a range-finding test, for which the key results only have been reported, a definitive limit test was conducted with a test concentration of 100 % saturated solution, a control group was also included. The test was conducted under semi-static test conditions with media preparation at 0 and 24 hours.

At the start of the test, five juvenile Daphnia magna were added to each test vessel. The Daphnia magna in each test vessel were observed at 24 and 48 hours. The Daphnia magna were not fed during the test.

For the 100 % saturated solution test concentration, analysis of the freshly prepared media at 0 hours showed the measured concentration to be 1.25 mg/L, the old media at 24 hours showed a concentration <LOQ (0.05 mg/L). New media analysed at 24 hours showed measured a concentration of 0.158 mg/L, the old media at 48 hours showed a measured concentration <LOQ (0.05 mg/L). The variation in achieved concentrations at 0 and 24 hours 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-globules, micelles passing through filters.

Due to the reduction in concentration observed over the 24-hour renewal periods it was considered justifiable to base concentration on time weighted mean measured concentrations; this was calculated to be 0.233 mg/L (Dissolved Fraction).

The validity criteria for control immobility (=10 %) and dissolved oxygen (=3 mg/L) were both satisfied. The test is therefore considered valid.

Under the conditions of the study the 48-hour EL50 was determined to be >100 mg/L and the corresponding NOEC was determined to be 100 mg/L based on the nomainl loading rate.

Description of key information

Under the conditions of the study the 48-hour LC50 was determined to be >100 mg/L and the corresponding NOEC was determined to be 100 mg/L based on the nomainl loading rate.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

The toxicity of the test material to the freshwater invertebrate, Daphnia magna, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 202. 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).

Based on the results of a range-finding test, for which the key results only have been reported, a definitive limit test was conducted with a test concentration of 100 % saturated solution, a control group was also included. The test was conducted under semi-static test conditions with media preparation at 0 and 24 hours.

At the start of the test, five juvenile Daphnia magna were added to each test vessel. The Daphnia magna in each test vessel were observed at 24 and 48 hours. The Daphnia magna were not fed during the test.

For the 100 % saturated solution test concentration, analysis of the freshly prepared media at 0 hours showed the measured concentration to be 1.25 mg/L, the old media at 24 hours showed a concentration <LOQ (0.05 mg/L). New media analysed at 24 hours showed measured a concentration of 0.158 mg/L, the old media at 48 hours showed a measured concentration <LOQ (0.05 mg/L). The variation in achieved concentrations at 0 and 24 hours 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-globules, micelles passing through filters.

Due to the reduction in concentration observed over the 24-hour renewal periods it was considered justifiable to base concentration on time weighted mean measured concentrations; this was calculated to be 0.233 mg/L (Dissolved Fraction).

The validity criteria for control immobility (=10 %) and dissolved oxygen (=3 mg/L) were both satisfied. The test is therefore considered valid.

Under the conditions of the study the 48-hour LC50 was determined to be >100 mg/L and the corresponding NOEC was determined to be 100 mg/L.