4,4'-(2,3-epoxypropoxy)-2,2'- dimethyl-5,5'-tert-butyldiphenylsulphide

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:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Analytical measurements were performed at the controls and at the applied test concentration levels at the beginning and at the end of the test.
- The samples were analysed by an HPLC.

Vehicle:

no

Details on test solutions:

- Because the test material is very 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. A saturated test material stock solution (nominally 100 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 test. This solution was stirred for about 48 hours and then the non-dissolved test material was removed by filtration through a fine (pore size: 0.22 μm) filter to give the saturated solution.
- To demonstrate that every effort was made to achieve a detectable concentration, an additional test concentration above the solubility level of the test material was tested parallel with the limit test concentration. Organic solvent (acetone) was used during the formulation procedure. An amount of 250 mg test material was dissolved in 50 mL acetone thereafter 0.1 mL from this stock solution was diluted in 1000 mL of OECD medium to give the test concentration of 0.5 mg/L (nominal).

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:

22.3 – 22.8 °C

pH:

7.54 – 9.06

Nominal and measured concentrations:

Nominal: 0.5 mg/L and 100 % saturated solution
The calculated geometric mean concentration of the 0.5 mg/L nominal concentration was 0.13 mg/L.
The concentration of test material was below the analytically detectable range in the 100 %saturated solution.

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 8193 lux (equivalent to 111 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 24, 48 and 72 hours) 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 test concentration and three in the control group. Because the test material is very poorly soluble in the test medium, the test concentrations were prepared using acetone as organic solvent (above the solubility level of the test material).
- Test concentrations: 0.125, 0.25 and 0.5 mg/L
- Results used to determine the conditions for the definitive study: Because significant toxicity was not observed during the preliminary concentration range-finding test, only one test concentration (at the solubility level of the test material in the test medium) and one control group were tested in a limit test.
Furthermore a test concentration (nominally 0.5 mg/L) using acetone as solvent and one additional control contained the solvent substance at the same concentration (0.1 mL/L) as that used in this test group were tested parallel with the limit test concentration.

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 were 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 – t1) + [(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

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.13 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.:

> 0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.13 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.:

0.13 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.:

> 0.13 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 68.50 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 10.30%.
- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.84%.
- All validity criteria were met, therefore the study can be considered as valid.

CONCENTRATIONS OF THE TEST MATERIAL
- The test material was not detected in the control samples (neither normal nor solvent control).
- The concentration of test material was below the analytically detectable range in the 100% saturated solution.
- Measured concentration in the test group of 0.5 mg/L (nominal) was 0.33 mg/L at the start and below the Limit of Detection (LOD = 0.05 mg/L) at the end of the experiment. In order to calculate the mean exposure concentration at this concentration level, the end concentration was taken as the Limit of Detection. The calculated geometric mean concentration was 0.13 mg/L.
- Biological results are related to the geometric mean of the measured test material concentrations.

CELL NUMBERS
- The cell number in each flask was determined at the 24th, 48th and 72nd hours.

AVERAGE SPECIFIC GROWTH RATES
- The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h average specific growth rates were not statistically significantly different from the control values at the used test groups.

AREAS UNDER THE GROWTH CURVES
- The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h areas were not statistically significantly different from the control values at the used test groups.

YIELD
- The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h yield values were not statistically significantly different from the control values at the used test groups.

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.
- 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 controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni 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 Bonferroni t-Test.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, no toxic effect was observed at both tested concentration levels. As the concentration of 0.5 mg/L (nominally) represented a higher level than the limit of solubility, it can be stated that the test material had no toxic effect at saturation (0.13 mg/L, measured).

Executive summary:

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

Significant toxic response was not observed during the preliminary concentration range-finding test therefore a limit test was performed in the main experiment using only one test concentration at the limit of solubility of the test material in the test medium (100% saturated solution) and one control group.

The measured concentrations were below the analytically detectable range in the case of 100% saturated solution. Furthermore a test concentration of 0.5 mg/L nominally (in the analytically measurable range) using acetone as solvent and one additional control contained the solvent substance at the same concentration (0.1 mL/L) as that used in the test group were tested parallel. The corresponding calculated concentration of this test group (based on the analytical measurements) was 0.13 mg/L. As the measured concentration deviated more than 20 percent from the nominal concentration during the test, the results are based on the measured geometric mean test material concentration.

The test design included six replicates at the test concentrations and six for the controls. Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni 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.

All validity criteria were met during this study.

The observed endpoints for the effect of the test material were: 72 h EbC50 value (biomass): > 0.13 mg/L (measured), 72 h ErC50 value (growth rate): > 0.13 mg/L (measured), 72h EyC50 value (yield): > 0.13 mg/L (measured), No-Observed Effect Concentration (NOEC): 0.13 mg/L (measured) and Lowest Observed Effect Concentration (LOEC): > 0.13 mg/L (measured).

Under the conditions of this study, no toxic effect was observed at both tested concentration levels. As the concentration of 0.5 mg/L (nominally) represented a higher level than the limit of solubility, it can be stated that the test material had no toxic effect at saturation (0.13 mg/L, measured).

Description of key information

Under the conditions of this study, no toxic effect was observed at both tested concentration levels. As the concentration of 0.5 mg/L (nominally) represented a higher level than the limit of solubility, it can be stated that the test material had no toxic effect at saturation (0.13 mg/L, measured).

Additional information

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

Significant toxic response was not observed during the preliminary concentration range-finding test therefore a limit test was performed in the main experiment using only one test concentration at the limit of solubility of the test material in the test medium (100% saturated solution) and one control group.

The measured concentrations were below the analytically detectable range in the case of 100% saturated solution. Furthermore a test concentration of 0.5 mg/L nominally (in the analytically measurable range) using acetone as solvent and one additional control contained the solvent substance at the same concentration (0.1 mL/L) as that used in the test group were tested parallel. The corresponding calculated concentration of this test group (based on the analytical measurements) was 0.13 mg/L. As the measured concentration deviated more than 20 percent from the nominal concentration during the test, the results are based on the measured geometric mean test material concentration.

The test design included six replicates at the test concentrations and six for the controls. Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni 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.

All validity criteria were met during this study.

The observed endpoints for the effect of the test material were: 72 h EbC50 value (biomass): > 0.13 mg/L (measured), 72 h ErC50 value (growth rate): > 0.13 mg/L (measured), 72h EyC50 value (yield): > 0.13 mg/L (measured), No-Observed Effect Concentration (NOEC): 0.13 mg/L (measured) and Lowest Observed Effect Concentration (LOEC): > 0.13 mg/L (measured).

Under the conditions of this study, no toxic effect was observed at both tested concentration levels. As the concentration of 0.5 mg/L (nominally) represented a higher level than the limit of solubility, it can be stated that the test material had no toxic effect at saturation (0.13 mg/L, measured).