2,2'-[(1-methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]bisoxirane

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:

02 April 2018 to 05 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- After the exposure period, the infrared absorption spectrum of the test material was measured. The spectrum obtained was consistent with that measured before the study, indicating that the test material was stable under the storage conditions.
- The purity of the test material is 93 %. The test was performed without correction by the purity and the toxicity data were obtained. The toxicity data corrected by the purity was reported.

Analytical monitoring:

yes

Details on sampling:

- The concentrations of the test material in all test solutions and test cultures were measured at the beginning of exposure and at 24, 48 and 72 hours after the beginning of exposure by Liquid Chromatography/Mass Spectrometry (LC/MS).

Vehicle:

no

Details on test solutions:

The test material was added to the dilution water, and then stirred for 48 hours. This solution was filtrated with a vacuum pump with a 0.45 µm filter to remove insoluble test material, and then used as the stock solution of test material. The test solutions were prepared from the stock solution diluted with dilution water.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: ATCC22662
- Source: American Type Culture Collection
- Algae have been maintained by period subculture using Gorham medium. Algae are kept under sterile condition. Sterile test has been performed periodically, every six months, to confirm that the algae are not contaminated.

ACCLIMATION
- Preculture period: From 30 March to 2 April 2018
- In order to adapt the algae to the test conditions and ensure that the algae were in the exponential growth phase when used to inoculate the test solutions, the algae were precultured under the same conditions as the test culture. Neither modified nor unusual cells were confirmed.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 ± 2 °C

pH:

7.8

Nominal and measured concentrations:

Nominal: 1.0, 2.2, 4.8, 10, 23, 50 and 110 mg/L
Corrected by the purity: 0.930, 2.05, 4.46, 9.30, 21.4, 46.5 and 102 mg/L
Geometric mean measured: 0.143, 0.287, 0.580, 1.37, 3.29, 8.21 and 16.5 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flask with aluminium cap
- Fill volume: 100 mL/vessel (99 mL/vessel when algae were inoculated to the test solution. 1 mL/vessel was used for analysis before inoculation)
- Aeration: open system with shaking (100 rpm)
- Initial cells density: 5×10^3 cells/mL, algae of exponential growth phase (dried weight of algae at the exponential growth phase: 1.8×10^−8 mg/cell, n=26)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium composition: NH4Cl 15 mg/L, MgCl2.6H2O 12 mg/L, CaCl2.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeCl3.6H2O 0.064 mg/L, Na2EDTA.2H2O 0.1 mg/L, H3BO3 0.185 mg/L, MnCl2.4H2O 0.415 mg/L, ZnCl2 0.003 mg/L, CoCl2.6H2O 0.0015 mg/L, CuCl2.2H2O 0.00001 mg/L, Na2MoO4.2H2O 0.007 mg/L and NaHCO3 50 mg/L.
The test guideline shows that the pH of the medium which is obtained at equilibrium between the carbonate system of the medium and the partial pressure of CO2 in atmospheric air is 8.1.
- Intervals of water quality measurement: The pH values of the test solutions and the test cultures were measured at the beginning of exposure and at 72 hours after the beginning of exposure. At the beginning of exposure, a part of the test solution in the extra vessel was collected and pH was measured. At 72 hours after the beginning of exposure, pH of the test culture was measured in one vessel in each test group. During the exposure period, temperatures, light intensities, and revolutions in the culturing apparatus were measured at least once a day.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination with white fluorescent lamp (on the surface of test culture)
- Light intensity and quality: 65 to 75 μE/m²/s

EFFECT PARAMETERS MEASURED:
- Measurement of biomass: The biomass of algae in each test vessel was measured at 24, 48 and 72 hours after the beginning of exposure. One millilitre of test culture (0.5 mL at 72 hours) was suspended in 9.0 mL of electrolyte (9.5 mL at 72 hours) to count the algal cells by electronic particle counter.
- Colour observation of test culture and microscopic observation of algae: The colour of the test cultures was observed by comparison with the extra vessel of each test group at the beginning of exposure and at 24, 48 and 72 hours after the beginning of exposure. At 72 hours after the beginning of exposure, appearance of algae was observed through a microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations: 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: Yes

EVALUATION OF THE RESULTS
- Calculation of Results
Growth curves: The mean values of the biomass for the control and each concentration group were plotted against time to produce plots of growth curves. Based on the plots of the control group, the exponential growth of the algae during the exposure period was checked.

Calculation of the growth rate and the percent inhibition of growth rate: The growth rate (μi-j) for exponentially growing algae was calculated according to the following equation:
µi-j = (ln Xj – ln Xi) / (tj – ti)
μi-j: growth rate from time i to j (/day)
Xi: biomass (cells/mL) at time ti (using the nominally inoculated biomass for X0)
Xj: biomass (cells/mL) at time tj
ti: time of ith measurement (/day)
tj: time of jth measurement (/day)
The percent inhibition (Iμ) of the growth rate in each concentration group was calculated by subtraction of the average specific growth rate (μt) for the treatment replicate from the mean value (μc) for the average rate in the control group according to the following equation:
Iµ = [(µc - µt) / µc] x 100
μc: mean value for average specific growth rate (μ) in the control group
μt: average specific growth rate for the treatment replicate

The percent inhibition (Iy) of the yields based on comparison
Yield was calculated according to the following equation:
Y = Nn - N0
Y: Yield
N0: Biomass at the start of the exposure (nominal value)
Nn: Biomass at tn hours

The percent inhibition (Iy) of the growth rate for each concentration group was calculated as the difference between the biomass of each replicate in that concentration group and the average yield in the control group according to the following equation:
Iy = ((Yc-Yt)/Yc) x100
Yc: average yield in the control group
Yt: yield in each replicate of the concentration group

Concentration of the test material for calculating inhibition concentration: The results were calculated from the geometric mean of measured concentrations corrected by the purity of test material. The geometric mean was calculated according to the following equation:
Mean Concentration = antilog[(1/(2(tn-t1)))∑[(log(conci)+log(conci+1)).(ti+1-ti)]]
Mean concentration = geometric mean
ti= initial time < t2 < ... tn = Final time
conci = initial concentration, conc2, ..., concn = final concentration

Calculation of 50 % growth inhibition concentration (EC50): Using growth inhibition rate Iμ (0-3d) from the beginning of exposure to 72 hours after the beginning of exposure, the concentration of the test material inhibiting 50 % growth of the algae (50 % growth inhibition concentration, EC50) was determined. Expressed as: Based on lµ values: ErC50 (0-72 h) Based on Iy values: EyC50 (0-72 h).

No Observed Effect Concentration (NOEC): Growth rates μ (0-3 d) of each concentration group were compared with the control group and the highest test concentration that indicated no significant difference, the geometric mean of the concentration group was taken as the No Observed Effect Concentration (NOEC: Expressed as NOECr (0-72h) and NOECy (0-72h)).

VALIDITY OF THE TEST
Validity of the test was assessed according to the following criteria:
The biomass in the control cultures should have increased by a factor of at least 16 within the exposure period.
The mean coefficient of variation for section by section daily specific growth rates in the control cultures must not exceed 35% through the exposure period.
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 16.5 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.21 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

RESULTS AND DISCUSSION
There were no significant circumstances that may have affected the reliability of the test results.

Conditions in the Culturing Apparatus, pH of Test Solutions and Test Cultures, and Appearance of Test Solutions:
The temperature in the culturing apparatus during exposure period ranged within 22 ± 2 °C, nominal value. The pH value of test solutions at the beginning of exposure was all 7.8 and test cultures at 72 hours after the beginning of exposure were 7.6 to 7.7. Test solutions before inoculation were colourless, and showed no suspended solids, no floating solids, and no precipitates in all test groups.

Concentrations of Test Material in Test Solutions and Test Cultures:
Measured concentrations of test material at the beginning of exposure in concentration group 1 to 7 were 0.160, 0.324, 0.633, 1.44, 3.48, 8.76 and 18.1 mg/L, respectively, and those at 72 hours after the beginning of exposure were 0.127, 0.259, 0.539, 1.31, 3.07, 7.79 and 15.8 mg/L, respectively.
The geometric means of measured concentrations of the test material in concentration group 1 to 7 were 0.143, 0.287, 0.580, 1.37, 3.29, 8.21 and 16.5 mg/L, respectively.

50% Growth Inhibition Concentration (EC50) and No Observed Effect Concentration (NOEC):
It was confirmed that algae grew exponentially in the control group.
50% growth inhibition concentration, ErC50 (0-72h): > 16.5 mg/L (95 % confidence limits: not available)
No observed effect concentration, NOECr (0-72h): 8.21 mg/L
50% growth inhibition concentration, EyC50 (0-72h): > 16.5 mg/L (95 % confidence limits: not available)
No observed effect concentration, NOECy (0-72h): 8.21 mg/L
The highest concentration of the test was 110 mg/L (Concentration corrected by the purity of test material: 102 mg/L, Geometric mean of measured concentration: 16.5 mg/L) of the maximum test concentration given in the Test Guideline. As the inhibition rate at the highest concentration was less than 50 %, the ErC50 and EyC50 values were shown as the range, "> maximum concentration".
The NOECr and NOECy values were determined by an analysis of variance (ANOV A), Williams test, subsequent to Bartlett test for homogeneity of variances. The significance level for all tests was set at a=0.05, except Bartlett test, which was set at a=0.01.

Observation of Algae:
In all test groups, the colour of the test cultures (as observed with the naked eye) showed a tendency to get more greenish during the exposure period.
By microscopic observation at the end of exposure, in all concentration groups neither unusual cell shape of algae (contraction, expansion, damaged cell etc.) nor agglutination was observed, and the algae looked normal compared with those in the control group.

Validity of the Test
The biomass in the control cultures increased exponentially above 16 fold during 72-hour culture. The mean coefficient of variation for section by section specific growth rates in the control cultures and the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 35 and 7%, respectively.
This test is valid because these results met the test validity criteria.

Results with reference substance (positive control):

ErC50 of the latest reference substance test = 0.809 mg/L (95% confidence limits: 0.800-0.818 mg/L,Exposure period: From December 18 to 21, 2017)
The average values of ErC50 since June, 2000 were as follows: Average ErC50 ± standard deviation = 0.810 ± 0.0694 mg/L, n=36, (Minimum-maximum = 0.687-0.965 mg/L)

Reported statistics and error estimates:

The NOECr value was determined by an analysis of variance (ANOVA), Williams test, subsequent to Bartlett test for homogeneity of variances. The significance level for all tests was set at α = 0.05, except Bartlett test, which was set at α = 0.01.

Table 1: Summary of Results

Nominal conc. (Nominal Conc. Corrected by Purity) [Mean measured conc.](mg/L)	Mean Growth Rate		Mean Biomass (cells/mL)
	Rate µ (0-3 d)	Inhibition (%) Iµ (0-3 d)	0 Hour	24 Hours	48 Hours	72 Hours
Control	1.90	-	5000	31500	231000	1500000
1.0 (0.930) [0.143]	1.94	-2.2	5000	28700	230000	1690000
2.2 (2.05) [0.287]	1.89	0.3	5000	29300	198000	1480000
4.8 (4.46) [0.580]	1.88	0.9	5000	28600	200000	1440000
10 (9.30) [1.37]	1.86	2.4	5000	26500	179000	1310000
23 (21.4) [3.29]	1.87	1.8	5000	26400	179000	1370000
50 (46.5) [8.21]	1.87	1.7	5000	29200	210000	1360000
110 (102) [16.5]	1.79**	5.7	5000	22900	154000	1080000

** Indicates a significant difference (α = 0.01) from the control.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the 50 % growth inhibition concentration, ErC50 (0-72h) was >16.5 mg/L and the no observed effect concentration, NOECr (0-72 h) was 8.21 mg/L.

Executive summary:

The toxicity of the test material to aquatic algae was investigated in accordance with the standardised guideline OECD 201, under GLP conditions.

Pseudokirchneriella subcapitata (unicellular green algae) were exposed to the test material for 72 hours in a static open system with shaking. Nominal test material concentrations of 1.0, 2.2, 4.8, 10, 23, 50 and 110 mg/L were selected based on the results of a rang-finding test. The test material concentrations were analysed by liquid chromatography/mass spectrometry (LC/MS).

Measured concentrations of test material at the beginning of exposure in concentration group 1 to 7 were 0.160, 0.324, 0.633, 1.44, 3.48, 8.76 and 18.1 mg/L, respectively, and those at 72 hours after the beginning of exposure were 0.127, 0.259, 0.539, 1.31, 3.07, 7.79 and 15.8 mg/L, respectively. The geometric means of measured concentrations of the test material in concentration group 1 to 7 were 0.143, 0.287, 0.580, 1.37, 3.29, 8.21 and 16.5 mg/L, respectively.

By microscopic observation at the end of exposure, in the concentration groups neither unusual cell shape of algae (contraction, expansion, damaged cell etc.) nor agglutination was observed, and the algae looked normal compared with that in the control group.

Under the conditions of this study the 50 % growth inhibition concentration, ErC50 (0-72h) was >16.5 mg/L and the no observed effect concentration, NOECr (0-72 h) was 8.21 mg/L.

Description of key information

Under the conditions of this study the 50 % growth inhibition concentration, ErC50 (0-72h) was >16.5 mg/L and the no observed effect concentration, NOECr (0-72 h) was 8.21 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

8.21 mg/L

Additional information

The toxicity of the test material to aquatic algae was investigated in accordance with the standardised guideline OECD 201, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Pseudokirchneriella subcapitata (unicellular green algae) were exposed to the test material for 72 hours in a static open system with shaking. Nominal test material concentrations of 1.0, 2.2, 4.8, 10, 23, 50 and 110 mg/L were selected based on the results of a rang-finding test. The test material concentrations were analysed by liquid chromatography/mass spectrometry (LC/MS).

Measured concentrations of test material at the beginning of exposure in concentration group 1 to 7 were 0.160, 0.324, 0.633, 1.44, 3.48, 8.76 and 18.1 mg/L, respectively, and those at 72 hours after the beginning of exposure were 0.127, 0.259, 0.539, 1.31, 3.07, 7.79 and 15.8 mg/L, respectively. The geometric means of measured concentrations of the test material in concentration group 1 to 7 were 0.143, 0.287, 0.580, 1.37, 3.29, 8.21 and 16.5 mg/L, respectively.

By microscopic observation at the end of exposure, in the concentration groups neither unusual cell shape of algae (contraction, expansion, damaged cell etc.) nor agglutination was observed, and the algae looked normal compared with that in the control group.

Under the conditions of this study the 50 % growth inhibition concentration, ErC50 (0-72h) was >16.5 mg/L and the no observed effect concentration, NOECr (0-72 h) was 8.21 mg/L.