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

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:

2015-05-22 - 2015-06-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

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:

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 allowed to thaw, then were diluted 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 study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.
The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 10 and 100 mg/L for a period of 72 hours. Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively.
For the definitive test a nominal amount of test item (200 mg) was added to the surface of 2 liters of culture medium to give the 100 mg/L loading rate. After the addition of the test item, the culture medium 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. Microscopic 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). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 ml discarded). Further filtration through Postlip filter paper was performed twice to ensure all undissolved test item was removed.
An aliquot (1 liter) of the WAF was inoculated with algal suspension (6.0 mL) to give the required test concentration of 100 mg/L loading rate WAF.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (green algae)
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): not mentioned

ACCLIMATION
- Acclimation period: not mentioned
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: no

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period described.

Hardness:

Hardness is not reported.

Test temperature:

24 ± 1 °C

pH:

6.7 - 7.6

Dissolved oxygen:

No details available.

Salinity:

Not applicable.

Nominal and measured concentrations:

Range-finding study: 10 and 100 mg/L (nominal), WAF
Definitive study: 0 (control), 100 mg/L (nominal), WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed, plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: glass, 100 mL of test preparation
- Aeration: constant
- Initial cells density: 103 cells/mL (range finding), 8.30 x 105 cells per mL (definitive test)
- Control end cells density: approximately 104 - 105 cells/mL (range finding), 5 x 103 cells per mL (definitive test)
- No. of vessels per concentration (replicates): 2 (range finding), 6 (definitive test)
- No. of vessels per control (replicates): 2 (range finding), 6 (definitive test)

OBSERVATION PERIODS
- at 0, 24, 48 and 72 hours

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
NaNO3: 25.5 mg/L
MgCl2.6H2O: 12.16 mg/L
CaCl2.2H2O: 4.41 mg/l
MgSO4.7H2O: 14.6 mg/L
K2HPO4: 1.044 mg/L
NaHCO3: 15 mg/L
H3BO3: 0.186 mg/L
MnCl2.4H2O: 0.415 mg/L
ZnCl2: 0.00327 mg/L
FeCl3.6H2O: 0.160 mg/L
CoCl2.6H2O: 0.00143 mg/L
Na2MoO4.2H2O: 0.00726 mg/L
CuCl2.2H2O: 0.000012 mg/L
Na2EDTA.2H2O: 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with O.1N NaOH or HCl.
- Intervals of water quality measurement: at 0 and 72 h

OTHER TEST CONDITIONS
- Sterile test conditions: not specified
- Adjustment of pH: yes, to pH 7.5
- Photoperiod: constant illumination
- Light intensity and quality: approximately 7000 lux provided by warm white lighting (380 - 730 nm)
- Temperature: 21± 1 °C master culture, 24 ± 1 °C for test
and constantly shaken at approximately 150 rpm for 72 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (Coulter® Multisizer Particle Counter)

TEST CONCENTRATIONS
- Test concentrations: 10 und 100 mg/L nominal WAF (range finding stuidy) und 100 mg/L nominal WAF (main test).
0.19 mg/L = LOQ
- Results used to determine the conditions for the definitive study: EL50 > 100mg/L nominal WAF

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities
- Unusual cell shape: no
- Unusual cell size: no

Results with reference substance (positive control):

- Results with reference substance valid?: Yes

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 - 72 h): 1.2 mg/L; 95% confidence limits 1.1 - 1.4 mg/L
EyCs50 (0 - 72 h): 0.63 mg/L; 95% confidence limits 0.57 - 0. 70 mg/L

No Observed Effect Concentration (NOEC) based on growth rate:0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate:1.0 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield:0.50 mg/L

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

Reported statistics and error estimates:

Statistical analysis of the growth rate and yield data was carried out for the control and 100 mg/L loading rate WAF test group using a Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing
several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P≥0.05), between the control and 100 mg/L loading rate WAF test group.

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 W AF was prepared using a stirring period of 95 hours followed by a 1-Hour settlement period.

Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.

The results showed no effect on growth rate at 10 and 100 mg/L loading rate WAF.

Based on this information a single loading rate of six replicates of 100 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.

Chemical analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 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 0.19 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.

Table 1: Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate (mg/L)		Cell Densities* (cells per mL)		Inhibition Values(%)
		0Hours	72Hours	Growth Rate	Yield
Control	R1	5.14E+03	7.89E+05	-	-
	R2	5.50E+03	9.93E+05
	Mean	5.32E+03	8.91E+05
10	R1	4.50E+03	8.25E+05	[3]	5
	R2	4.29E+03	8.72E+05
	Mean	4.39E+03	8.49E+05
100	R1	5.34E+03	6.94E+05	3	20
	R2	4.86E+03	7.26E+05
	Mean	5.10E+03	7.10E+05

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1 and R2 = Replicates 1 and 2

[Increase in growth compared to controls ]

Definitive Test

Chemical Analysis of Test Loading Rates

Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 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 0.19 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration ofless than the LOQ.

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.

The measured concentrations obtained from both the range-finding and definitive test were significantly less than those obtained from the validation of mixing period trial. Upon inspection of the validation data it became apparent that not all steps were taken to ensure that maximum quantity of undissolved test item was removed from the aqueous phase as was practically possible. As such it was considered that the higher measured test concentrations obtained from the validation work was due to the presence of undissolved test item in the sample.

Growth Data

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

The mean cell densities versus time for the definitive test are presented in Figure 1.

From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-Hour exposure period.

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

Table 2: Cell Densities and pH Values in the Definitive Test

Nominal Loading Rate (mg/L)		pH	Cell Densities* (cells per mL)				pH
		0 h	0 h	24h	48h	72 h	72 h
Control	R1	7.6	4.72E+03	1.90E+04	1.50E+05	6.71E+05	6.7
	R2		5.47E+03	1.98E+04	1.47E+05	8.84E+05
	R3		4.83E+03	2.41E+04	1.97E+05	1.03E+06
	R4		4.96E+03	2.42E+04	1.85E+05	9.94E+05
	R5		5.10E+03	1.94E+04	1.76E+05	8.96E+05
	R6		4.46E+03	2.57E+04	1.70E+05	1.03E+06
	Mean		4.93E+03	2.20E+04	1.71E+05	9.18E+05
100	R1	7.5	5.76E+03	3.00E+04	2.33E+05	9.22E+05	7.0
	R2		4.88E+03	2.60E+04	1.90E+05	8.09E+05
	R3		4.93E+03	3.19E+04	2.23E+05	8.73E+05
	R4		4.96E+03	3.48E+04	2.27E+05	8.71E+05
	R5		5.58E+03	3.07E+04	2.32E+05	9.05E+05
	R6		5.41E+03	3.21E+04	2.35E+05	8.52E+05
	Mean		5.25E+03	3.09E+04	2.23E+05	8.72E+05

* Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1 - R; =Replicates 1 to 6

Table 3: Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/mL/hour)
		Day0 -1	Day1 - 2	Day2 - 3
Control	R1	0.056	0.086	0.062
	R2	0.057	0.084	0.075
	R3	0.065	0.088	0.069
	R4	0.066	0.085	0.070
	R5	0.056	0.092	0.068
	R6	0.068	0.079	0.075
	Mean	0.061	0.086	0.070

Table 4: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0-72h	%Inhibition	0-72h	%Inhibition*
Control	R1	0.068		6.67E+05
	R2	0.072		8.78E+05
	R3	0.074		1.03E+06
	R4	0.074	-	9.89E+05	-
	R5	0.072		8.91E+05
	R6	0.074		1.03E+06
	Mean	0.072		9.13E+05
	SD	0.002		1.38E+05
100	R1	0.072	0	9.16E+05
	R2	0.071	1	8.04E+05
	R3	0.072	0	8.68E+05
	R4	0.072	0	8.66E+05
	R5	0.072	0	9.00E+05
	R6	0.071	1	8.46E+05
	Mean	0.072	0	8.67E+05	5
	SD	0.001		3.95E+04

* In accordance with the OECD test guideline only the mean value for yield is calculated R1 - ~ = Replicates 1 to 6

SD = Standard Deviation

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 186 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours: 4.93 x 103 cells per mL

Mean cell density of control at 72 hours: 9.18 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 18% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 - 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Validity criteria fulfilled:

yes

Remarks:

Validity criteria of the applied guideline were met.

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green algae Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guideline 201 referenced as EU Method C.3 and is compliant with GLP criteria.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method which was determined to be 0.19 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.

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. Exposure of Pseudokirchneriella subcapitata to the test item gave ErL₅₀ values and EbL₅₀ of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate referring to growth rate and yield (biomass) was 100 mg/L loading rate WAF.

No effects (size differences, unusual cell shapes) were noted during the test.

Description of key information

A study (Vryenhoef, 2016) was performed to assess the effect of the test item on the growth of the green algae Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guideline 201 referenced as EU Method C.3 and was performed in accordance with GLP.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method which was determined to be 0.19 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.

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. Exposure of Pseudokirchneriella subcapitata to the test item gave ErL₅₀ values and EbL₅₀ of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate referring to growth rate and yield (biomass) was 100 mg/L loading rate WAF.

No effects (size differences, unusual cell shapes) were noted during the test.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L