1-O-β-(2 ,3-di-O-alka(e)noyl-6 -O-acetyl-D-mannopyranosyl)-D-erythritol

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:

19 December 2018 - 21 February 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was conducted according to OECD Guideline 202 and in accordance with the OECD Principles of GLP. All relevant validity criteria were met. The method used to determine ‘water solubility’ in the OECD 202 study was by use of a water accommodated fraction followed by quantitative HPLC analysis. This is not considered appropriate methodology for determining the ‘solubility’ of a surface-active substance such as MEL-B. As the test substance is surface active, a surface tension study was performed to determine the critical micelle concentration instead. This was determined to be 5.15 mg/L as described in Section 4.8 and should be considered as the greatest aqueous concentration that can be reliably tested as per the Reach R7b endpoint guidance. This means that any material added to an aqueous system at a concentration greater than 5.15 mg/L will result in the formation of micelles, which are not considered to be in solution. This also means that during the daphnia study, the organisms at any concentration greater than 5.15 mg/L were exposed to a mixture of dissolved MEL-B and MEL-B micelles. This brings into question the validity of results obtained from any group tested above 5.15 mg/L as for the purpose of aqueous toxicity testing, substances must be fully in solution. It should be considered that the tested concentrations under the CMC remain valid, but the concentrations above the CMC are invalid, for the reasons stated above. Therefore, as the 48 h EC50 value originally calculated under the study (2.58 mg/L) has been generated from concentration data under the CMC, this can be considered a relatively robust value. The study report originally estimates the 24 h EC50 values as > 46.2 mg/L, however, as this concentration cannot have been reliably tested, this has been revised in the summary to > 5.15 mg/L, the CMC, as no effects were noted and as this is the maximum bioavailable amount of test item. The full data has been reported, but this endpoint have been revised in light of the CMC value.

Qualifier:

according to guideline

Guideline:

other: Daphnia sp. Acute Immobilisation Test

Version / remarks:

"Daphnia sp. Acute Immobilization Test" stipulated in the "Testing Methods for New Chemical Substances " (March 31, 2011, No.0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29, 2011, No.5, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No.110331009, Environmental Policy Bureau, Ministry of the Environment, Japan; latest revision, March 29, 2018, No.0329-13, Pharmaceutica1 and Food Safety Bureau, Ministry of Health, Labour and Welfare; No.20180326-1, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No.1803293, Environmental Policy Bureau, Ministry of the Environment, Japan)

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 13, 2004

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document, No 23 (Second Edition), July 2018 " Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals"

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TOYOBO CO., LTD,

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test sample was stored in a cold and dark storage place.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.5, 1.58, 5, 15.8 and 50 mg/L (geometric series with a factor of 3.16 - square root of 10)
- Sampling method: At the start of exposure (from another solution sampled separately from the preparation container), and before renewal, after renewal, and end of exposure (the mixed solution taken out with equal volume from the middle layer of the test solution in the test vessels) (4 occasions in total). Approximately 100 mL of sample taken on each occasion.
- Sample storage conditions before analysis: not reported

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The weighed test sample and dilution water were mixed to produce each nominal test concentration. They were stirred with a magnetic stirrer for 48 h at about 20°C. Then the suspension was filtered with a membrane filter (GV, 0.22 µm pore size, Merck Millipore) with suction. The filtrate was stirred for 30 minutes at about 20°C to recover dissolved oxygen concentration decreased by the suction filtration. This was carried out for the start of exposure and renewal of test solution.
- Eluate: dechlorinated tap water

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Clone A
- Age at study initiation (mean and range, SD): less than 24 h old
- Method of breeding: not reported
- Source: University of Sheffield, UK, 1990
- Age of parental stock (mean and range, SD): 21 days
- Feeding during test: none


ACCLIMATION
- Acclimation period: Young daphnids produced by parents that were cultured in the test facility were used
- Acclimation conditions (same as test or not): The parents were bred in the same quality of water, water temperature (20±1°C) and photoperiod (16 h light, 8 h dark)
- Type and amount of food: parent animals were fed Chlorella vulgaris algae at equivalent of 0.1-0.2 mgC/day/daphnia.
- Feeding frequency: daily
- Health during acclimation (any mortality observed): 0% mortality

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

20 mg/L (chemical characteristics of dilution water conducted on 10 July 2018)

Test temperature:

19.8-19.9°C

pH:

7.8-8.0

Dissolved oxygen:

8.6-9.0 mg/L

Salinity:

-

Conductivity:

8.7 mS/m (chemical characteristics of dilution water conducted on 10 July 2018)

Nominal and measured concentrations:

0.500, 1.58, 5, 15.8, 50 mg/L nominal concentrations; 0.25, 0.79, 3.64, 13.1, 46.2 mg/L measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beaker
- Type (delete if not applicable): covered with transparent plastic lid
- Volume of solution: 100 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water
- Total organic carbon: <0.5 mg/L
- Chlorine: less than 0.02 mg/L
- Alkalinity: 21 mg/L
- Conductivity: 8.7 mS/m
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light, 8 h dark
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : yes, 24h 48 h

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 0.5, 1, 5, 50, 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 5.15 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

refer to justification

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.58 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: none
- Observations on body length and weight: none
- Other biological observations: none
- Mortality of control: none
- Other adverse effects control: none
- Immobilisation of control: none
- Abnormal responses: 14 dapnhids immobilised, 13 displaying lethargy and 2 showing reduced activity at 48 h in 3.64 mg/L measured concentration. 7 daphnids immobilised, 1 displaying lethargy and 2 showing reduced activity at 24 h at 13.1 mg/L measured concentration, 20 daphnids showing immobilisation and lethargy at 48 h.
3 daphnids immobilised, 1 displaying lethargy and 2 showing reduced activity at 24 h at 46.2 mg/L measured concentration, 20 daphnids showing immobilisation and lethargy at 48 h.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? Test with reference substance conducted periodically (9-11 Oct 2018)
- ECx: 48 h EC50 0.13 mg/L

Reported statistics and error estimates:

The 24 h and 48 h EC50 values for Daphnia magna were >3.64 mg/L and 2.58 mg/L, determined using Microsoft Excel, using the binomial test method (US EPA/600/485/013, March 1985). No confidence limits were provided.

Table 1: Immobility

 

Measured Concentration^a (mg/L)	Vessel	24 hours		48 hours
		Number of immobilised daphnids/Total daphnids	Immobility (%)	Number of immobilised daphnids/Total daphnids	Immobility (%)
Control	A	0/5	0	0/5	0
	B	0/5		0/5
	C	0/5		0/5
	D	0/5		0/5
0.250 (reference value)	A	0/5	0	0/5	0
	B	0/5		0/5
	C	0/5		0/5
	D	0/5		0/5
0.790	A	0/5	0	0/5	0
	B	0/5		0/5
	C	0/5		0/5
	D	0/5		0/5
3.64	A	0/5	0	4/5	70
	B	0/5		3/5
	C	0/5		5/5
	D	0/5		2/5
13.1	A	3/5	35	5/5	100
	B	1/5		5/5
	C	1/5		5/5
	D	2/5		5/5
46.2	A	1/5	15	5/5	100
	B	2/5		5/5
	C	0/5		5/5
	D	0/5		5/5

_{a: Time weighted mean of the measured concentrations}

 

Table 2: Measured concentrations of test item in test solutions

 

Nominal Concentration (mg/L)	Measured Concentration (mg/L) (Percentage of measured concentration versus that at each preparation %
	At the Start	24 hours		At the End	Time-weighted arithmetic mean
		Before the Renewal	After the Renewal
Control	n.d	n.d	n.d	n.d	-
0.500	n.d	n.d	n.d	n.d	0.250^a
1.58	0.859	0.576 (67.0)	1.05	0.717 (68.5)	0.790
5.00	3.81	3.31 (87.0)	4.04	3.44 (85.1)	3.64
15.8	13.9	12.8 (92.4)	13.0	12.7 (97.4)	13.1
50.0	47.3	45.2 (95.5)	46.5	45.9 (98.9)	46.2

_{a: reference value calculated from the geometric rate and the measured concentration of the lowest quantified exposure level.}

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour EC50 of the test item to D. magna was estimated to be 2.58 mg/L.

Executive summary:

A study was performed according to OECD 202 (2004) to assess the acute toxicity of the test item to Daphnia magna. Following a range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to aqueous solutions of the test item at nominal concentrations of 0.50, 1.58, 5.0, 15.8 and 50.0 mg/L for 48 h under semi-static conditions. Chemical analysis showed measured test concentrations to range from 0.576 to 47.3 mg/L, although levels in the nominal 0.50 mg/L concentration were below the limit of quantification (0.5 mg/L). The time-weighted arithmetic mean measured concentrations were determined to be 0.250 (reference value), 0.790, 3.64, 13.1 and 46.2 mg/L. The critical micelle concentration of the test item was determined to be 5.15 mg/L, and therefore this brings into question the validity of results obtained from any group tested above 5.15 mg/L. The 48 h EC50 value originally calculated under the study (2.58 mg/L) has been generated from concentration data under the CMC value and is a robust value. The study report originally estimates the 24 h EC50 values as > 46.2 mg/L, however, as this concentration cannot have been reliably tested, this has been revised in the summary to > 5.15 mg/L, the CMC values, as no effects were noted, and this is the maximum bioavailable amount of the test item. This is in line with ECHA Guidance document R.7b, Table 7.8 -3 (2017).  

Therefore, exposure of Daphnia magna to the test item gave the following result based upon time-weighted average concentrations: 48-h EC50– 2.58 mg/L. All validity criteria were met.

Description of key information

48h EC₅₀ 2.58 mg/L; OECD 202; Adachi R (2019)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.58 mg/L

Additional information

A study was performed according to OECD 202 (2004) to assess the acute toxicity of the test item to Daphnia magna. Following a range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to aqueous solutions of the test item at nominal concentrations of 0.50, 1.58, 5.0, 15.8 and 50.0 mg/L for 48 h under semi-static conditions. Chemical analysis showed measured test concentrations to range from 0.576 to 47.3 mg/L, although levels in the nominal 0.50 mg/L concentration were below the limit of quantification (0.5 mg/L). The time-weighted arithmetic mean measured concentrations were determined to be 0.250 (reference value), 0.790, 3.64, 13.1 and 46.2 mg/L.The critical micelle concentration of the test item was determined to be 5.15 mg/L, and therefore this brings into question the validity of results obtained from any group tested above 5.15 mg/L. The 48 h EC50 value originally calculated under the study (2.58 mg/L) has been generated from concentration data under the CMC value and is a robust value. The study report originally estimates the 24 h EC50 values as > 46.2 mg/L, however, as this concentration cannot have been reliably tested and would have contained solids as well as the soluble bioavailable fraction, this value has been revised in the summary to > 5.15 mg/L. This is the CMC value, at which no effects were observed i.e. the maximum bioavailable amount of the test item. Hence, the test item will not be toxic to the 50 % level during this period. This is in line with ECHA Guidance document R.7b, Table 7.8 -3 (2017).

Therefore, exposure of Daphnia magna to the test item gave the following result based upon time-weighted average concentrations: 48-h EC₅₀– 2.58 mg/L. All validity criteria. were met.