Oxirane, 2,2'-[methylenebis[(2,6-dimethyl-4,1-phenylene)oxymethylene]]bis-

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:

18 April 2011 to 21 April 2011

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidelines for studies on the new chemical substance as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law)

Version / remarks:

1973, amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint ordinance of The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- The concentration of the test material in the test solution was determined at the beginning and at the end of the study.
- On each occasion six replicate samples were taken from the test solution and one sample was taken from the control solution.
- The samples were analysed by HPLC using UV detection.

Vehicle:

no

Details on test solutions:

Because the test material is poorly soluble in water, in order to achieve the maximum solubility level of the test material (limit test concentration), a test solution was prepared using a ‘saturated solution method’. The test material was crushed to a powder (to achieve better dissolution) and then a supersaturated test material stock solution (nominal loading rate of 1000 mg/L) was prepared by dispersing/dissolving the amount of test material into the test medium (OECD Medium) two days before the start of the study (on day -2). This solution was shaken for about 48 hours and then the non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the saturated solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of LAB Research Ltd.
- Method of cultivation: The pre-culture was intended to give an amount of alga suspension suitable for the inoculation of test cultures. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal Growth Medium to 10^7 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.6 – 23.2 °C

pH:

7.63 – 8.99

Nominal and measured concentrations:

Nominal: 100% v/v saturated solution
Measured geometric mean: 0.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.
- fill volume: 100 mL
- Initial cells density: The initial cell number in the test cultures was 10^4 cells/mL.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests. Separate stock solutions were first prepared in deionised water. The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations:
Stock solution 1 (macro nutrients): NH4Cl 15.0 mg/L, MgCl2.6H2O 12.0 mg/L, CaCl2.2H2O 18.0 mg/L, MgSO4.7H2O 15.0 mg/L and KH2PO4 1.6 mg/L.
Stock solution 2 (iron): FeCl3.6H2O 64.0 μg/L and Na2EDTA.2H2O 100.0 μg/L.
Stock solution 3 (trace elements): H3BO3 185.0 μg/L, MnCl2.4H2O 415.0 μg/L, ZnCl2 3.0 μg/L, CoCl2.6H2O 1.5 μg/L, CuCl2.2H2O 0.01 μg/L and Na2MoO4.2H2O 7.0 μg/L.
Stock solution 4 (bicarbonate): NaHCO3 50.0 mg/L
- Intervals of water quality measurement: Culture temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The pH was checked at the beginning and at the end of the study, in the control and each concentration.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 8097 lux (equivalent to 109 μE/m²/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15% and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED:
- The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
- Microscopic observation of the algal cells at the test concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
Range finding study
- A concentration range-finding test was conducted to determine the approximate toxicity of the test material so that appropriate test concentrations can be selected for use in the definitive test. Algal cells were exposed to each concentration of the test material plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three in the control group.
- Test concentrations: 1, 10, 50 and 100% v/v saturated solutions
- Results used to determine the conditions for the definitive study: Because no toxic effect was observed in the preliminary range-finding test, only one test concentration at the solubility level of the test material in the test medium (100% v/v saturated solution) and one control group were tested in a limit test.

CALCULATIONS
- Calculation of Average Specific Growth Rate:
Concentration-effect relationship was calculated by comparing growth rates in control, test cultures in the following way. The average specific growth rate (μ) for individual cultures are calculated from the following relationship:

µ = [ln(Nn) - ln(N0)] / tn – t0

Where
ln (Nn) = natural logarithm of measured number of cells/mL at time tn
ln (N0) = natural logarithm of measured number of cells/mL at time t0
t0 = time (hour) of the beginning of the test
tn = time (hour) of nth measurements after the beginning of the test

The percentage inhibition of growth rate = % Iµ:

% Iµ= [(µc - µt) / µc ] ·100 %

Where
% Iμ = percent inhibition in average specific growth rate
μc = mean growth rate of the control
μt = mean growth rate of test concentration t

- Calculation of Area Under the Growth Curve:

A = [(N1 – N0) / 2] · t1 + [(N1 + N2 – 2N0) / 2] · (t2 – 21) + [(Nn-1 + Nn – 2N0) / 2] · (tn – tn – 1)

Where
N0 = nominal number of cells/mL at time t0 (start of the test)
N1 = mean measured number of cells/mL at t1 (24 hours)
N2 = mean measured number of cells/mL at t2 (48 hours)
Nn = mean measured number of cells/mL at tn
t1 = time of first measurement after start of the test
t2 = time of second measurement after start of the test
tn = time of nth measurement after start of the test

The percentage inhibition of area = % IA

% IA = [(Ac – At) / Ac] · 100 %

Where % IA = percent inhibition in area under the growth curve
Ac = mean area of the control
At = mean area of test concentration t

- Calculation of Yield:
Yield is calculated as the biomass at the end of the test minus the starting biomass for each single vessel of controls and treatments. For each test concentration and control, mean yield values were calculated.

Percentage inhibition in yield = % Iy

% Iy = [(yc – yi) / yc] · 100

Where:
yc = mean value for yield in the control group
yi = mean value for yield for the test concentration

Area under the growth curve (biomass), average specific growth rate and yield were calculated for each test flask. Then the mean area under the growth curve, the growth rate and mean yield were determined as arithmetic mean value over all test flasks per treatment.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

biomass and yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

biomass and yield

Details on results:

VALIDITY
- The cell density in the control cultures increased by a factor of 69.33 within three days.
- The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 7.26%.
- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.13%.
- All validity criteria were met, therefore the study can be considered as valid.

AVERAGE SPECIFIC GROWTH RATES
- The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h average specific growth rate was not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 0.6 mg/L (measured).

AREAS UNDER THE GROWTH CURVES
- The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h areas were not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 0.6 mg/L (measured).

YIELD
- The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h yield was not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 0.6 mg/L (measured).

Results with reference substance (positive control):

The 72h ErC 50: 1.40 mg/L, (95% confidence limits: 1.29 – 1.53 mg/L)
The 72h EbC 50: 0.92 mg/L, (95% confidence limits: 0.85 – 0.99 mg/L)
The 72h EyC 50: 0.92 mg/L, (95% confidence limits: 0.85 – 1.00 mg/L)

Reported statistics and error estimates:

- The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
- The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
- The ErC50, EbC50 and EyC50 values of the test material were determined from the raw data.
- Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software.
- For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by 2 Sample t-Test (α = 0.05) by TOXSTAT software.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 72 h ErC50 for the unicellular green alga, Pseudokirchneriella subcapitata, was determined to be >0.6 mg/L. The 72-hour No-Observed Effect Concentration (NOEC) was determined to be 0.6 mg/L. The results show that the test material had no toxic effect at saturation; the EC50 results and the LOEC were higher than the solubility level of the test material in the test medium.

Executive summary:

The toxicity of the test material to the unicellular green alga, Pseudokirchneriella subcapitata, was investigated in accordance with the standardised guidelines OECD 201, EU Method C.3., OPPTS 850.5400 and Japanese guidelines, under GLP conditions.

Because no significant inhibition of algal growth was observed during the preliminary range-finding test, a limit test was carried out using only one concentration at the solubility level of the test material in the test medium (100% v/v saturated solution) and one control group.

The concentration of test material was analytically determined during the experiment. The measured concentration was 0.8 mg/L at the start and 0.5 mg/L at the end of the test. The geometric mean measured concentration was determined to be 0.6 mg/L.

The test design included six replicates at test concentration and six replicates for the untreated control. Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software. The ErC50, EbC50 and EyC50 values of the test material were determined directly from the raw data (because of the lack of toxic effects).

Under the conditions of this study, the observed endpoints for the effect of the test material (measured concentration) on algal growth inhibition in terms of biomass, growth and yield were: 72 h EC50 >0.6 mg/L, the No-Observed Effect Concentration (NOEC) = 0.6 mg/L and the Lowest Observed Effect Concentration (LOEC) > 0.6 mg/L. The test material had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test material in the test medium.

Description of key information

Under the conditions of the study, the 72 h ErC50 for the unicellular green alga, Pseudokirchneriella subcapitata, was determined to be >0.6 mg/L. The 72-hour No-Observed Effect Concentration (NOEC) was determined to be 0.6 mg/L. The results show that the test material had no toxic effect at saturation; the EC50 results and the LOEC were higher than the solubility level of the test material in the test medium.

Additional information

The toxicity of the test material to the unicellular green alga, Pseudokirchneriella subcapitata, was investigated in accordance with the standardised guidelines OECD 201, EU Method C.3., OPPTS 850.5400 and Japanese guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Because no significant inhibition of algal growth was observed during the preliminary range-finding test, a limit test was carried out using only one concentration at the solubility level of the test material in the test medium (100% v/v saturated solution) and one control group.

The concentration of test material was analytically determined during the experiment. The measured concentration was 0.8 mg/L at the start and 0.5 mg/L at the end of the test. The geometric mean measured concentration was determined to be 0.6 mg/L.

The test design included six replicates at test concentration and six replicates for the untreated control. Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software. The ErC50, EbC50 and EyC50 values of the test material were determined directly from the raw data (because of the lack of toxic effects).

Under the conditions of the study, the observed endpoints for the effect of the test material (measured concentration) on algal growth inhibition in terms of biomass, growth and yield were: 72 h EC50 >0.6 mg/L, the No-Observed Effect Concentration (NOEC) = 0.6 mg/L and the Lowest Observed Effect Concentration (LOEC) > 0.6 mg/L. The test material had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test material in the test medium.