Reaction product of D-Glucopyranoside, methyl; esterified with oleic acid, methyl ester

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:

2015-07-23 - 2015-08-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of The United Kingdom

Analytical monitoring:

not specified

Details on sampling:

Preparation of Calibration Standards
The test item (nominal 100 mg) was dissolved in tetrahydrofuran (100 mL) to prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further diluted with diluent* to produce a solution of 10 mg/L. Defined volumes of this solution were then diluted with diluent* to obtain calibration standards in the range of 0.10 to 1.0 mg/L. A second series of calibration standards was similarly prepared in the range of 0.25 to 1.5 mg/L. These standards were used to determine the recovery and test sample concentrations.

Preparation of Linearity Standards
The test item (nominal 100 mg) was dissolved in tetrahydrofuran (100 mL) to prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further diluted with diluent* to produce a solution of 10 mg/L. Defined volumes of this solution were then diluted with diluent* to obtain calibration standards in the range of 0.10 to 1.0 mg/L. A second series of calibration standards was similarly prepared in the range of 0.25 to 1.5 mg/L. These standards were used to evaluate the linearity of the analytical system.

Preparation of Spiked Recovery Samples
To demonstrate the validity of the analytical procedure, volumes of test medium were spiked with the test item and the recovery was assessed. The test item (nominal 100 mg) was initially dissolved in tetrahydrofuran to prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further diluted with tetrahydrofuran to produce a stock solution of 250 mg/L. A defined volume of this stock solution was diluted with test medium to obtain spiked recovery
samples at a concentration of 0.5 mg/L. Five replicates were prepared and subjected to the same treatment as the test samples. In addition, test medium without the addition of the test item (synthetic control) was also analyzed.

Preparation of Test Samples
The test samples were thawed with the aid of sonication, prior to diluting with tetrahydrofuran.

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.
Prior to addition of the test item a glass siphon tube was placed in the test media. A nominal amount of test item (2200 mg) was added to the surface of 22 liters of test water to give the 100 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. The stirring was stopped after 95 hours and the mixture allowed to stand for 1 hour. Observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length) and filtering through postlip filter papers. A length of Tygon tubing was attached to the top of the glass siphon tube and a glass wool plug was inserted into the opposite end of the tubing. The WAF was removed by mid-depth siphoning (the first 75-100 mL discarded). This was then filtered through two sheets of postlip filter paper to give the 100 mg/L loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 72 and 96 hours

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
- Age at study initiation (mean and range, SD): juvenile
- Length at study initiation (length definition, mean, range and SD): 4.8 cm (sd = 0.52)
- Weight at study initiation (mean and range, SD): 1.39 g (sd = 0.46)
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods
- Type and amount of food during the test: Fishes received no food during exposure.

ACCLIMATION
- Acclimation period: 2015-07-20 - 2015-07-27
- Acclimation conditions (same as test or not): yes
- Type and amount of food during acclimation:commercial trout pellets which were discontinued 24 hours prior to the start of the definitive test
- Feeding frequency during acclimation: not mentioned
- Health during acclimation (any mortality observed): zero mortality in the 7 days prior to the start of the test

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period described.

Hardness:

approximately 140 mg/L as CaCO3

Test temperature:

14 - 15 °C

pH:

7.7 - 10

Dissolved oxygen:

8.2 - 10.1 mg O2/L

Salinity:

not applicable

Nominal and measured concentrations:

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 than
the threshold loading rate.
Definitive test: 100 mg/L loading rate WAF (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 25 - 30 liter vessels
- Type: closed (vessels were covered)
- Material, size, headspace, fill volume: glass, containing 20 litres of media
- Aeration: via narrow bore glass tubes.
- Renewal rate of test solution (frequency): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 7
- Biomass loading rate: 0.49 g bodyweight/liter

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener). After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: 0.996 mg/L
- Pesticides: < 0.007 µg/L
- Chlorine: 0.248 mg/L
- Conductivity: 411.711 µS/cm
- Culture medium different from test medium: same
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test.

OTHER TEST CONDITIONS
- Adjustment of pH: approximately 7.4
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition period

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any mortalities and sub-lethal effects of exposure were recorded at 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.

TEST CONCENTRATIONS
100 mg/L nominal WAF loading rate

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

mortality (fish)

Details on results:

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

Results with reference substance (positive control):

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 greater than 60% of ASV (6.2 mg O2/L) in the control and test vessels.

Reported statistics and error estimates:

No statistics were reported.

Sublethal observations / clinical signs:

Validation of Mixing Period

Preliminary investigational work indicated that there was a significant increase in the amount of dissolved test item when the preparation period was extended from 24 to 96 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 95 hours followed by a 1-Hour settlement period.

The results are summarized as follows:

Nominal Loading Rate (mg/L)	Time (Hours)
	24	96
	Measured Concentration (mg/L)	Measured Concentration (mg/L)
100	14.9	39.9

Definitive Test

Chemical Analysis of Test Loading Rates

Chemical analysis of the test preparations at 0, 24, 72 and 96 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.068 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or 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.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LL50 values based on the nominal test concentrations of greater than 100 mg/L. The No Observed Effect Loading Rate was 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Table 1: Cumulative Mortality Data in the Definitive Test

Nominal Loading Rate (mg/L)	Cumulative Mortality (Initial Population = 7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0

Sub-Lethal Effects

There were no sub-lethal effects of exposure observed in 7 fish exposed to a 100 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 greater than 60% of ASV ( 6.2 mg O₂/L) in the control and test vessels.

Water Quality Criteria

The results of the water quality measurements are given in Table 2. Temperature was maintained at 14 °C to 15 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Table 2: Water Quality Measurements (attached)

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 100 mg/L loading rate was observed to be a clear colorless to slightly yellow water column with globules of test item dispersed throughout the water column and at the surface of the water. After 95 hours stirring and a 1-Hour standing period the 100 mg/L loading rate was were observed to be a clear colorless to cloudy water column with test item remaining dispersed throughout the water column and/or at the surface of the water. Observation of the WAF showed a cloudy dispersion of test item throughout the water column and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length) and subsequent filtration through two postlip filter papersheets. During the test the 100 mg/L loading rate was observed to be a slightly cloudy to cloudy homogenous dispersion.

Validity criteria fulfilled:

yes

Remarks:

Validity criteria of the applied guideline were fulfilled.

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated using the threshold approach and gave a 96-Hour LL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Executive summary:

The acute toxicity of the test substance towards rainbow trout (Oncorhynchus mykiss) was determined according to OECD Guideline 203 and EU Method C.1 in compliance with GLP.

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 than the threshold loading rate.

Therefore seven fish were exposed to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of approximately 14°C 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 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

The 96-Hour LL₅₀ (Lethal Loading rate) based on nominal loading rates was greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Description of key information

The acute toxicity of the test substance towards towards rainbow trout (Oncorhynchus mykiss) was determined according to OECD Guideline 203 and EU Method C.1 in compliance with GLP (Sacker, 2015(a)).

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 than the threshold loading rate.

Therefore seven fish were exposed to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of approximately 14°C 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 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

The 96-Hour LL₅₀ (Lethal Loading rate) based on nominal loading rates was greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L