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

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:

2015-06-11 - 2015-07-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

In the range-finding test the dilution water used was Elendt M7. As the daphnia are cultured in Elendt M7 this was considered not to affect the results of the test. Exposure of organisms to a Water Accommodated Fraction (WAF) of the test item.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

In the range-finding test the dilution water used was Elendt M7. As the daphnia are cultured in Elendt M7 this was considered not to affect the results of the test. Exposure of organisms to a Water Accommodated Fraction (WAF) of the test item.

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of The United Kingdom

Analytical monitoring:

no

Details on sampling:

The test item concentration in the test samples was determined by high performance liquid chromatography with mass spectrometry (HPLC-MS) using an external standard. The test item gave a chromatographic profile consisting of several unresolved peaks.
The method was developed by the Department of Analytical Services, Envigo Research Limited, Shardlow, UK.

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 solutions 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.

* Diluent: tetrahydrofuran: test medium (50:50 v/v)

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, twenty daphnids ( 4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 to 22 °C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: 1st instar
- Source: in-house laboratory cultures
- Age at study initiation (mean and range, SD): less than 24 hours
- Feeding: no food supply during exposure

- Other: Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C.The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods

ACCLIMATION
- Type and amount of food: Each culture was fed with a suspension of algae.(Desmodesmus subspicatus) and Tetramin® flake food suspension
- Feeding frequency: daily

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Post exposure observation period:

No post exposure observation period described.

Hardness:

approximately 250 mg/Las CaCO3

Test temperature:

21 to 22 °C

pH:

7.8 to 9.0

Dissolved oxygen:

8.4 to 9 mg O2/L

Salinity:

Not applicable

Nominal and measured concentrations:

Preliminary range-finding test: 1.0, 10 and 100 mg/L nominal loading rate WAF
Definitive test: 100 mg/L nominal loading rate WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass beakers
- Type (delete if not applicable): closed (covered)
- Material, size, headspace, fill volume: 100 mL test media
- Aeration: none
- No. of organisms per vessel: 10 (range-finding study), 5 (definitive test)
- No. of vessels per concentration (replicates): 1 (range finding), 4 (definitive test)
- No. of vessels per control (replicates): 1 (range finding), 4 (definitive test)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7 medium for range-finding test, econstituted water (ISO medium) for definitive test
Reconstituted Water - Elendt M7 Medium
H3B03: 0.715 mg/L
MnCl2.4H2O: 0,090 mg/L
Li Cl: 0.077 mg/L
RbCl: 0.018 mg/L
SrCl2.6H2O: 0.038 mg/L
NaBr: 0.004 mg/L
Na2MoO4.2H2O: 0.016 mg/L
CuCl2.2H2O: 0.004 mg/L
Zn Cl2: 0.013 mg/L
CoCl2.6H2O: 0.010 mg/L
KI: 0.0033 mg/L
Na2SeO3: 0.0022 mg/L
NH4VO3: 0.00058 mg/L
Na2EDTA.2H2O: 0.625 mg/L
FeSO4.7H2O: 0.249 mg/L
CaCl2.2H2O: 293.8 mg/L
NaHCO3: 64.8 mg/L
MgSO4.7H2O: 123.3 mg/L
Na2SiO3.9H2O: 10 mg/L
KCl: 5.8 mg/L
NaNO3: 0.274 mg/L
K2HPO4: 0.184 mg/L
KH2PO4: 0.143 mg/L
Thiamine hydrochloride: 0.075 mg/L
Cyanocobalamine (vitamin B12): 0.0010 mg/L
D(+) biotin (vitamin H): 0.00075 mg/L

Reconstituted water - ISO medium
CaCl2.2H2O: 294 mg/L
MgSO4.7H2O: 123 mg/L
NaHCO3: 65 mg/L
KCL: 5.8 mg/L
total hardness: 250 mg/L CaCO3
- Culture medium different from test medium: Elendt M7 medium for range-finding test, reconstituted water (ISO medium) for definitive test
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: 7.9 +/- 0.3 at 21 °C (range-finding), 7.8 +/- 0.2 mg/L (definitive test)
- Photoperiod: 16 hrs light / 8 hours dark, with 20 minute dawn and dusk transition periods.
- Light intensity: 200 - 1200 lux (626 - 657 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mobility

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L loading rate (WAFs)
- Results used to determine the conditions for the definitive study: No immobilization was observed at 1.0, 10 and 100 mg/L loading rate WAF. Limit test at 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

24 h

Dose descriptor:

EL50

Remarks:

WAF

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL50

Remarks:

WAF

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No sub-lethal effects or immobility of exposure were observed throughout the test
- Observations on body length and weight: No sub-lethal effects of exposure were observed throughout the test
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no

Results with reference substance (positive control):

- Valid reference substance used
- Concentrations: 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.:
- EC50: 1.1 mg/L (SD-not possible to determine) after 24 hours, NOEC: 0.56 mg/L
- EC50: 0.75 mg/L (SD 0.69 - 0.82) after 48 hours, NOEC: 0.56 mg/L
- Other: The results from the positive control with potassium dichromate were within the normal range for this reference material.

Reported statistics and error estimates:

An estimate of the EL50 values was given by inspection of the immobilization data.

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 23 to 95 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.

 

Table 1: Preliminary investigational work on the influence of the duration of stirring on the amount of dissolved test item

Nominal Loading Rate (mg/L)	Time (Hours)
	23	95
	Measured Concentration (mg/L)	Measured Concentration (mg/L)
100	14.9	39.9

 

Range-finding Test

Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 2.

No immobilization was observed at 1.0, 10 and 100 mg/L loading rate WAF.

Based on this information, a single loading rate of four replicates, of 100 mg/L, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilization or adverse reactions to exposure were observed.

Chemical analysis of the 100 mg/L loading rate WAF test preparation at 0 hours (Table 3) showed a measured test concentration of 1.2 mg/L. There was a significant decline in the measured concentration at 48 hours to 0.50 mg/L indicating that the test item was not stable under test conditions.

Table 2: Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal Loading Rate (mg/L)	Observations (Initial Population: 10 Per Replicate)
	24 Hours		48 Hours
	Cumulative Immobilized Daphnia	Observations	Cumulative Immobilized Daphnia	Observations
Control	0	10N	0	10N
1.0	0	10N	0	10N
10	0	10N	0	10N
100	0	10N	0	10N

 

Table 3: Results for Range-Finding Samples

Timepoint [hours]	Nominal Loading RateCnom [mg/L]	Measured Concentration of Test Item in Sample Vial x [mg/L]	Sample Preparation Factor F	Determined Concentration of Test Item in Range-Finding Sample c [mg/L]
0	100	0.600	2	1.20
48	100	0.251	2	0.502

 

Definitive Test

 

Chemical Analysis of Test Loading Rates

Chemical analysis of the fresh test preparation at 0 hours (Table 4) showed the measured test concentration to be 0.19 mg/L. Chemical analysis of the aged test preparation at 48 hours showed a measured test concentration of less than the limit of quantification (LOQ) of the analytical method employed was obtained which was determined to be 0.043 mg/L.

The dissolved test item may have been one or several components of the test item. 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.

 

Table 4: Results for Test Samples

Timepoint [hours]	Nominal Loading Rate Cnom [mg/L]	Measured Concentration of Test Item in Sample Vial x [mg/L]	Sample Preparation Factor F	Determined Concentration of Test Item in Test Sample c [mg/L]
0	Control 100	<LOQ 0.0935	2 2	<LOQ 0.187
48	Control 100	<LOQ <LOQ	2 2	<LOQ <LOQ

LOQ=Limit of Quantification

For reporting purposes, x has been calculated from c retrospectively.

 

Immobilization Data

Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 5.

 

Table 5: Cumulative Immobilization Data and Observations in the Definitive Test (attached)

 

There was no immobilization in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours. Inspection of the immobilization data gave the following results:

 

Time (h)	EL50(mg/L Loading Rate WAF)	95% Confidence limits (mg/L Loading Rate WAF)
24	>100	n/a
48	>100	n/a

 

The No Observed Effect Loading rate after 48 hours exposure was 100 mg/L loading rate WAF.

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

 

Sub-Lethal Effects

No sub-lethal effects of exposure were observed throughout the test.

 

Validation Criteria

The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

 

Positive control

Analysis of the immobilization data by probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results:

Time Point [hours]	EC50 (mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
24	1.1	Not possible to determine	0.56	1.0
48	0.75	0.69         - 0.82	0.56	1.0

 

 

The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

 

Water Quality Criteria

The results of the water quality measurements are given in Table 6. Temperature was maintained at 21 to 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Throughout the test the light intensity was observed to be in the range 626 to 657 lux.

 

Table 6: Water Quality Measurements

Nominal Loading Rate (mg/L)	0 Hours			24 Hours	48 Hours
	pH	mg O2/L	T °C	T °C	pH	mg O2/L	T °C
Control R1	7.8	8.4	21	21	8.0	9.0	22
100 R1	7.8	8.4	21	21	7.9	8.8	22

 

Vortex Depth Measurements

The vortex depth was recorded at the start and end of the 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 clear colorless water column with globules of test item on the surface and dispersed throughout. After 95 hours stirring and a 1-Hour standing period the 100 mg/L loading rate was observed to be a clear colorless water column with globules of test item on the surface. Microscopic examination of the WAF showed micro-dispersions of test item to be present and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length).

Microscopic examination after filtering showed the glass wool plug had not removed all the micro dispersions and the WAF was further filtered through postlip filter paper.

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Validity criteria fulfilled:

yes

Remarks:

Validity criteria of the applied OECD Guideline are met.

Conclusions:

The study report describes a valid OECD guideline study conducted under certificated GLP compliance. The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated. Based on the low water solubility, Water Accommodated Fractions (WAFs) were prepared for testing. Immobilisation effects were recorded in the static freshwater test. The EL50(48h) was determined as > 100 mg/L loading rate WAF while the NOELR (48h) amounts to 100 mg/L.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Executive summary:

The acute toxicity of the test test towards Daphnia magna was determined according to OECD Guideline 202 and EU Method C.2 in compliance with GLP. Following a preliminary range-finding test, twenty daphnids (four replicates of five animals) were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L for 48 hours at a temperature of approximately 21 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

The EL₅₀ (48h) for the test material to Daphnia magna 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. The EC₅₀ (48h) for the reference material to Daphnia magna based on nominal concentrations was 0.75 mg/L with 95% confidence limits of 0.69 - 0.82 mg/L. The No Observed Effect Concentration was 0.56 mg/L. The results from the positive control with potassium dichromate were within the normal range for this reference item.

Description of key information

The acute toxicity of the test towards Daphnia magna was determined according to OECD Guideline 202 and EU Method C.2 in compliance with GLP (Sacker, 2015(b)). Following a preliminary range-finding test, twenty daphnids (four replicates of five animals) were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L for 48 hours at a temperature of approximately 21 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

The EL₅₀ (48h) for the test material to Daphnia magna 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. The EC₅₀ (48h) for the reference material to Daphnia magna based on nominal concentrations was 0.75 mg/L with 95% confidence limits of 0.69 - 0.82 mg/L. The No Observed Effect Concentration was 0.56 mg/L. The results from the positive control with potassium dichromate were within the normal range for this reference item.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L