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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 December 2018 - 25 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 201 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 201 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 algae 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 NOEC calculated under the study (0.298 mg/L) is well under the CMC, this is a scientifically valid value and the group can be considered to have been reliably tested. The study report originally estimates the EC50 as >93 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 close to 50% inhibition were noted at any of the tested concentrations, whether valid or invalid, and this is the maximum bioavailable amount of test item. The full data has been reported, but the final endpoints have been revised in light of the CMC value.

Qualifier:

according to guideline

Guideline:

other: Algal Growth Inhibition Test

Version / remarks:

"Algal Growth Inhibition 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)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006, Annex 5 corrected July 28, 2011

Deviations:

not specified

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: Control, 0.32, 1, 3.2, 10, 32, 100 mg/L
- Sampling method: 100 mL at 0 h (sampled from preparation containerat all test levels), 100 mL at each of 24 and 48 h (from test vessels at all test levels) and 90 mL at 72 h (from all test levels)
- 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 test sample and medium were mixed to prepare the nominal concentration, and they were stirred with a magnetic stirrer for 48 h. Then the suspension was filtered with a membrane filter (GV 0.22 µm pore size, Merck Millipore) by suction. The filter was used as the test solution and divided into each test vessel.
- Eluate: OECD Medium as per Guideline 201 - see Table 1
- Controls: untreated medium only
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the start of exposure, the appearance of test solution in the nominal 100, 32 and 10 mg/L concentrations was slightly white, depending on the concentration. The other test solutions were clear and colourless.
- Other relevant information: At the end of exposure, the appearance of the nominal 100 mg/L test solutions was 'whitish light green', in nominal 32 mg/L the appearance was 'light green' and the appearance in nominal 10 mg/L was ' a little light green'. The other concentrations were green due to algal growth.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection
- Age of inoculum (at test initiation): 3 day old preculture
- Method of cultivation: same as test conditions

ACCLIMATION
- Acclimation period: 3 day old pre-culture
- Culturing media and conditions (same as test or not): same as test conditions
- Any deformed or abnormal cells observed: none

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

-

Test temperature:

22.1-22.2

pH:

7.8-7.9

Dissolved oxygen:

-

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

0.32, 1, 3.2, 10, 32, 100 mg/L nominal concentrations; 0.0942, 0.298, 2.11, 8.01, 28.3, 93.0 mg/L geometric mean measured concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: sterilised 300 mL Erlenmeyer flask (with gas permeable Silicosen®)
- Material, size, headspace, fill volume: 100 mL test solution
- Initial cells density: 0.75 x 10E4 cells/mL
- Control end cells density: approx. 1.29 x 10E6 cells/mL, based upon 172 time increase in growth during test period.
- No. of organisms per vessel: 750000 cells per vessel
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes - as per OECD 201, see also table 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not reported
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals: not reported
- Pesticides: not reported
- Chlorine: not reported
- Alkalinity: not reported
- Ca/mg ratio: not reported
- Conductivity: not reported
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH measured at start and end of test, light intensity and temperature measured daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: LED Light providing nominal 90 µmol/m2/s in wavelength range 400-700 nm
- Incubator: Test conducted in incubator with temperature and illumination control, with continuous shaking.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] spectrophotofluorometer
- Chlorophyll measurement: yes, chlorophyll fluorescence value
- Other: Microscopic observation at end of test period

TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10 (approx 3.16)
- Range finding study
- Test concentrations: 0.3, 1, 5 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: see justification in adminitrative data

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.298 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 5.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): yes
- Unusual cell shape: no
- Colour differences: At the end of exposure, the appearance of the 93.0 mg/L test solutions was 'whitish light green', in the 28.3 mg/L solutions the appearance was 'light green' and the appearance in the 8.01 mg/L solutions was ' a little light green'. The other concentrations were green due to algal growth.
- Flocculation: not noted
- Adherence to test vessels:
- Aggregation of algal cells: Many aggregated cells were observed in the 93.0 mg/L solutions, some aggregated cells were observed in the 28.3 and 8.01 mg/L solutions. The conditions of the cells in the other solutions, including the controls, was normal.
- Any stimulation of growth found in any treatment: no
- 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? yes
- EC50: 1.2 mg/L
- Other: Test conducted with potassium dichromate, 22-25 October 2018

Reported statistics and error estimates:

Bartlett's test was done to determine homogeneity of variance for the data. Then a one-way ANOVA and Dunnett's multiple comparison test was used to determine the significant difference between the control and exposure levels. Statistical analysis for estimation of the EC50 was conducted using Microsoft Excel. The EC10 was estimated using the Probit method on SPSS v24 with a correction for heterogeneity violations and logaritmic transfromation to lead to a more relaible fit of data set.

Table 2 Biomass at each time

Measured Concentration (mg/L)	No.	Chlorophyll fluorescence value (relative unit)
		0 hour^b	24 hours	48 hours	72 hours
Control	A	32	210	1200	6000
	B	32	230	1200	5500^c
	C	32	230	1200	5600
	D	32	220	1100	5900
	E	32	230	1200	5900
	F	32	220	1200	5500^c
	Mean	32	220	1200	5700
	S.D	0	8.2	32	210
0.0942 (reference value)	A	32	210	1200	5900
	B	32	220	1200	6000
	C	32	210	1200	5600
	Mean	32	210	1200	5900
	S.D	0	5.5	16	220
0.298	A	32	210	1100	5600
	B	32	210	1200	5600
	C	32	210	1100	5600
	Mean	32	210	1200	5600
	S.D	0	1.6	41	16
2.11	A	32	200	1000	4800
	B	32	200	1100	5000
	C	32	200	1100	5100
	Mean	32	200	1100	5000
	S.D	0	2.4	38	180
8.01	A	32	170	820	3500
	B	32	200	880	4000
	C	32	170	860	3700
	Mean	32	180	860	3800
	S.D	0	15	29	220
28.3	A	32	170	720	3300
	B	32	170	700	3200
	C	32	160	660	3000
	Mean	32	160	700	3200
	S.D	0	4.9	28	170
93.0	A	32	150	610	2900
	B	32	140	630	2800
	C	32	160	700	3200
	Mean	32	150	650	3000
	S.D	0	6.7	48	180

b: value based upon measured value of pre-culture

c: minimum cell growth in the control (biomass at end of exposure/biomass at start of exposure) – 5500/32 = 172

Table 3 Growth Rate and Growth Rate Inhibition

 

Measured Concentration (mg/L)	No.	Growth Rate (0-3 d)	Growth Rate Inhibition (%)
Control	A	1.75	-
	B	1.72	-
	C	1.73	-
	D	1.74	-
	E	1.74	-
	F	1.72	-
	Mean	1.73	-
	S.D.	0.0120	-
0.0942	A	1.74	-0.67
	B	1.75	-1.0
	C	1.73	0.36
	Mean	1.74	-0.45
	S.D.	0.0125	0.72
0.298	A	1.73	0.37
	B	1.72	0.37
	C	1.73	0.28
	Mean	1.73	0.34
	S.D.	0.000943	0.054
2.11	A	1.67	3.6
	B	1.69	2.6
	C	1.69	2.2
	Mean	1.68	2.8
	S.D.	0.0123	0.71
8.01	A	1.57	9.2
	B	1.61	7.0
	C	1.59	8.3
	Mean	1.59	8.1
	S.D.	0.0196	1.1
28.3	A	1.55	11
	B	1.54	11
	C	1.51	13
	Mean	1.53	11
	S.D.	0.0178	1.0
93.0	A	1.51	13
	B	1.50	14
	C	1.53	11
	Mean	1.51	13
	S.D.	0.0203	1.2

 

Table 4 Variation of Growth Rates

 

Control No.	Growth Rate (0-3 d)	Standard Deviation	Coefficient of Variation (%)
A	1.75	0.121	6.9	11 (mean)
B	1.72	0.225	13
C	1.73	0.207	12
D	1.74	0.158	9.1
E	1.74	0.195	11
F	1.72	0.202	12
Average Specific Growth Rate (0-3 d)	1.73	-
Standard Deviation	0.0120
Coefficient of Variation (%)	0.69

Table 5 Measured Concentration of Test Item in Test Solutions

 

Nominal Concentration (mg/L)	Measured Concentration (mg/L) (Percentage of measured concentration versus that at the start %)
	Start	24 hours	48 hours	End	Geometric Mean
Control	n.d.	n.d.	n.d.	n.d.	-
0.32	n.d.	n.d.	n.d.	n.d.	0.0942^b
1	0.679	n.d.^a	n.d.^a	n.d.^a	0.298
3.2	2.68	2.14 (79.8)	2.22 (82.8)	1.45 (54.1)	2.11
10	8.56	8.22 (96.1)	7.87 (92.0)	7.38 (86.3)	8.01
32	30.1	28.4 (94.5)	27.9 (92.8)	27.0 (89.7)	28.3
100	96.7	92.5 (95.6)	93.2 (96.4)	90.0 (93.0)	93.0

n.d.: <0.506 mg/L

a: in accordance with OECD Guidance Document No. 23, half the determination limit of the test item (0.253 mg/L) was adopted for the calculation of geometric mean, because the test item was detected but not quantified

b: Reference value calculated from the geometric rate and the measured concentration of the lowest quantified exposure level (1.0 mg/L)

Validity criteria fulfilled:

yes

Remarks:

The test is considered valid as in the controls cell growth increased by a factor of at least 16 (actual 172), the mean CoV for section by section growth rates did not exceed 35% (actual 11%) and the CoV for specific growth rate was <7% (actual 0.69%)

Conclusions:

The 72-hour EC50 of the test item to P. subcapitata was estimated to be >5.15 mg/L. The NOEC was determined to be 0.298 mg/L.

Executive summary:

A study was performed according to OECD 201 (2006, corrected 2011) to assess the toxicity of the test item to green algae (Psuedokirchneriella subcapitata). Following a range finding study, algal cultures were exposed to aqueous solutions of the test item at nominal concentrations of 0.32, 1, 3.2, 10, 32 and 100 mg/L for 72 h under static test conditions, under constant illumination. Biomass was measured by means of a spectrophotofluorometer. Chemical analysis showed measured test concentrations to range from 0.679 to 96.7 mg/L, although several analyses in the nominal 1.0 mg/L concentration, and all analyses in the nominal 0.32 mg/L concentration were below the limit of quantification (0.506 mg/L). The geometric mean measured concentrations were determined to be 0.0942 (reference value), 0.298, 2.11, 8.01, 28.3 and 93.0 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 NOEC value originally calculated under the study (0.298 mg/L) has been generated from concentration data under the CMC value and is a robust value. The study report originally estimates the 72 h EC50 value as > 93.0 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 value, as no effects close to 50% inhibition were noted, and this is the maximum bioavailable amount of the test item. As only the the soluble fraction is bioavailable and this does not change above the CMC. This is in line with ECHA Guidance document R.7b, Table 7.8 -3 (2017).

The EC10 was also derived using SPSS v24, here the EC10 was estimated to be 28 mg/L, well above the CMC of the test item. As a precaution the EC10 was set to 5.15 mg/L, in line with ECHA Guidance document R.7b, Table 7.8-3 (2017).

Exposure of P. subcapitata to the test item gave the following results based upon the geometric mean measured concentrations: 72 h EC50(growth rate) > 5.15 mg/L, 72 hour EC10 (growth rate) > 5.15 mg/L, 72h NOEC (growth rate) 0.298 mg/L. All validity criteria for a valid test were met.

Description of key information

72 h EC50(growth rate) > 5.15 mg/L, 72h NOEC (growth rate) 0.298 mg/L; EC10 (growth rate) 5.15 mg/L; OECD 201; Ono M (2019)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

5.15 mg/L

Additional information

A study was performed according to OECD 201 (2006, corrected 2011) to assess the toxicity of the test item to green algae (Psuedokirchneriella subcapitata). Following a range finding study, algal cultures were exposed to aqueous solutions of the test item at nominal concentrations of 0.32, 1, 3.2, 10, 32 and 100 mg/L for 72 h under static test conditions, under constant illumination. Biomass was measured by means of a spectrophotofluorometer. Chemical analysis showed measured test concentrations to range from 0.679 to 96.7 mg/L, although several analyses in the nominal 1.0 mg/L concentration, and all analyses in the nominal 0.32 mg/L concentration were below the limit of quantification (0.506 mg/L). The geometric mean measured concentrations were determined to be 0.0942 (reference value), 0.298, 2.11, 8.01, 28.3 and 93.0 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 NOEC value originally calculated under the study (0.298 mg/L) has been generated from concentration data under the CMC value and is a robust value. The study report originally estimates the 72 h EC50 value as > 93.0 mg/L, however, as this concentration cannot have been reliably tested as solids and the soluble (bioavailable) fraction would have been present, this has been revised to > 5.15 mg/L. This is the CMC value. Further as no effects close to 50% inhibition were noted (max. 14 % inhibition), and as the maximal bioavailable fraction of the test item would have been present, the conclusion is considered robust. This is in line with ECHA Guidance document R.7b, Table 7.8 -3 (2017). The EC10 was also derived using SPSS v24, here the EC10 was estimated to be 28 mg/L, well above the CMC of the test item. As a precaution the EC10 was set to 5.15 mg/L, in line with ECHA Guidance document R.7b, Table 7.8-3 (2017).

Considering the above, exposure of P. subcapitata to the test item gave the following results based upon the geometric mean measured concentrations: 72 h EC50(growth rate) > 5.15 mg/L, 72h NOEC (growth rate) 0.298 mg/L. All validity criteria for a valid test were met.