(2S)-1-isopropoxy-1-oxopropan-2-yl pivalate

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:

2014-07-08 to 2014-07-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 201 with GLP certificate. All validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on June 03-05, 2013 / signed on November 05, 2013

Specific details on test material used for the study:

- Storage condition of test material: Keep in the dark at 0 - 10 °C, under nitrogen
- Stability under test conditions: Not specified, assumed to be stable

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via GC-MS at the start (0 hours) and at the end of the exposure (72 hours). At the start of the exposure samples were taken out of the additional replicates provided without algae. At the end of the exposure samples were taken out of the pooled test replicates per concentration level and control with algae.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Stock solution: 100 mg/L were freshly prepared with dilution water
- Dispersion treatment: Agitation
- Controls: Six replicates (without test item) were exposed under the same test conditions as the test vessels.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE/m2/s for 24 hours per day.
- Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

Dilution water medium has a nominal hardness of 0.24 mmol Ca+Mg/L

Test temperature:

22.5-23.5 °C (23.0 °C)

pH:

7.80-8.10 (t=0 h); 8.34-8.75 (t=72 h)

Dissolved oxygen:

None

Salinity:

None

Nominal and measured concentrations:

Nominal concentrations: 3.125, 6.25, 12.5, 25, 50 and 100 mg/L
Geometric mean measured concentrations: 2.14, 4.46, 8.38, 16.6, 35.4 and 67.9 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs
- Test volume: 109 mL
- Initial cells density: Nominal: approximately 5000-50000 cells/mL; Current: 7085 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Application: Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels.
- Incubation: The flasks were positioned randomly and repositioned daily.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the OECD guideline 201

OTHER TEST CONDITIONS
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Photoperiod: 24 hours/day light
- Light intensity and quality: 64.3 to 74.2 (70.8) µE/m2/s
- Light homogeneity: The light homogeneity over the incubation area was within -9.24 to 4.74 % of the average light intensity (required: within ± 15 % from the mean light intensity).

EFFECT PARAMETERS MEASURED:
- Chlorophyll a-fluorescence: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self fluorescence was found in the preliminary range finding test at the concentration level of 100 mg/L.
- Microscopic evaluation: Microscopic evaluation of the cells at the start and end of the incubation was carried out. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers or aggregation of algae cells.
Physical Chemical Parameters:
- The pH-value at the beginning of the exposure was measured in one additional replicate of each concentration level and the control. At the end of the exposure, it was measured from pooled replicates per concentration level and control, respectively. The room temperature was measured and recorded continuously. Light intensity was measured prior to the start of the test.

TEST CONCENTRATIONS
- Range finding study: In a non-GLP preliminary range finding test, three nominal test concentrations of 1, 10 and 100 mg/L were tested. Inhibition of growth rate was 1, 1 and 54% at test concentrations of 1, 10 and 100 mg/L, respectively. Respective inhibitions of yield were 6, 4 and 93%.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

16.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

35.4 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.:

27.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 24.9-30.3 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

45.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 40.8-50.6 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 67.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

16.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

35.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

15.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 12.8-18.7 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

18.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 15.8-22.1 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

30.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 25.1-36.8 mg/L

Details on results:

Biological data:
Microscopic evaluation of the cells at the start of the incubation period revealed no morphological abnormalities. At the end of incubation period, the algae of the concentration levels 35.4 and 67.9 mg/L were deformed (rounder and thickened). The other concentrations and the control showed no morphological abnormalities. All effect values are given based on geometric mean measured test item concentrations.

Measured Exposure Concentrations during the Definitive Test:
The concentrations of the test item ST 11 C 13 were determined at the start of the exposure (0 h) and at the end of the exposure (72 h) of all tested concentration levels and the control via GC-MS. The recovery rate of the test item ST 11 C 13 at the start of the exposure was in the range of 92 to 108 % of the nominal values at all tested concentration levels. The recovery rate at the end of the exposure was in the range of 45 to 49 % of the nominal values. All effect values are given based on the geometric mean measured concentration of the test item.

Results with reference substance (positive control):

Results with reference substance (potassium dichromate) valid
- The ErC50 value based on Growth Rate Inhibition: 0.661 mg/L (95% CI: 0.638-0.685 mg/L)
- The EyC50 value based on Yield Inhibition: 0.341 mg/L (95% CI: 0.286-0.377 mg/L)

Reported statistics and error estimates:

EC-values and statistical analyses: EC10, EC20 and EC50 values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC, LOEC and statistical analyses: The NOEC/LOEC were determined by calculation of statistically significant differences of growth rate and yield inhibition. As a standard, One Way Analysis of Variance (ANOVA) and Dunnett’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. The Shapiro-Wilk test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The α-value (acceptable probability of incorrectly concluding that there is a difference) is α = 0.05.

Table 6.1.5/1: Cell Densities

 

Geometric mean measured test item concentrations [mg/L]	Mean Cell Density [cells/mL]
	0 hours	24 hours	48 hours	72 hours
67.9	7085	18931	71689	406083
35.4	7085	20770	133236	750057
16.6	7085	29096	213213	1438248
8.38	7085	34161	295522	1917944
4.46	7085	36943	361519	2056897
2.14	7085	33201	361532	1824143
Control	7085	34811	292696	1702056

 

Table 6.1.5/2: Evaluation after 72 h             

 

Geometric mean measured test item concentrations (nominal test item concentrations) [mg/L]	Mean values
	Growth Rate [d^-1]		Inhibition of Growth Rate [%]	Yield [cells/mL]		Inhibition of Yield [%]
67.9	(+)	1.35	26	(+)	398998	76
35.4	(+)	1.55	15	(+)	742972	56
16.6	(-)	1.77	3	(-)	1431163	16
8.38	(-)	1.87	-2	(-)	1910859	-13
4.46	(-)	1.89	-4	(+)*	2049812	-21
2.14	(-)	1.85	-1	(-)	1817058	-7
Control		1.83			1694971

*significance caused by stimulation of growth

 

Validity Criteria

The study meets the validity criteria of the guideline:

Validity Criterion	Required	This study
Increase of the cell growth in the control cultures	Exponentially≥16-fold corresponding to a specific growth rate of 0.92 day^-1	240-fold (specific growth rate 1.83 day^-1)
Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures	<35 %	15.7 %
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	<7 %	2.51 %

Validity criteria fulfilled:

yes

Conclusions:

In this study, test item was found to inhibit the growth of the freshwater green algae Pseudokirchneriella subcapitata with the following effect values (geometric mean measured test item concentrations): The EC50 value with 95 % CI for inhibition of growth rate (ErC50) after 72 hours was > 67.9 mg/L mg/L. The EC10-value with 95 % CI for inhibition of growth rate (ErC10) after 72 hours was 27.5 (24.9 - 30.3) mg/L. The NOEC-value for inhibition of growth rate after 72 hours was 16.6 mg/L.

Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test material at nominal concentrations of 3.125, 6.25, 12.5, 25, 50 and 100 mg/L corresponding to the geometric mean measured concentrations of the test item: 2.14, 4.46, 8.38, 16.6, 35.4 and 67.9 mg/L for 72 h. The study was conducted under static conditions with an initial cell density of 7085 cells/mL. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. 

 

The concentrations of the test item ST 11 C 13 were analytically verified by GC-MS at the start and the end of the exposure of all test concentrations and the control. The recovery rate of the test item ST 11 C 13 at the start of the exposure was in the range of 92 to 108 % of the nominal values at all tested concentration levels. The recovery rate at the end of the exposure was in the range of 45 to 49 % of the nominal values. All effect values were based on the geometric mean measured concentrations of the test item ST 11 C 13.

 

All validity criteria of the test guideline were fulfilled (Increase of cell growth in the control cultures, mean coefficients of variation for specific growth rates and coefficient of variation of average specific growth rates).

 

In this study, test item was found to inhibit the growth of the freshwater green algae Pseudokirchneriella subcapitata with the following effect values (geometric mean measured test item concentrations): The EC50 values with 95 % CI for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 67.9 mg/L and 30.2 (25.1-36.8) mg/L, respectively. The EC10-values with 95 % CI for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 27.5 (24.9 - 30.3) mg/L and 15.7 (12.8-18.7) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 16.6 mg/L, respectively.

Description of key information

OECD Guideline 201, GLP, key study, validity 1:

72h-ErC50 (Pseudokirchneriella subcapitata) > 67.9 mg/L based on geometric mean measured concentration

72h-ErC10 (Pseudokirchneriella subcapitata) = 27.5 mg/L (95% CI: 24.9 - 30.3 mg/L) based on geometric mean measured concentration

72h-NOECr (Pseudokirchneriella subcapitata) = 16.6 mg/L based on geometric mean measured concentration

Key value for chemical safety assessment

EC50 for freshwater algae:

67.9 mg/L

EC10 or NOEC for freshwater algae:

16.6 mg/L

Additional information

One key study is available (NOACK, 2014) to assess the influence of the registered substance on the growth of the freshwater green algae species Pseudokirchneriella subcapitata in a 72h static condition, according to OECD Guideline 201 with GLP statement. The algae were exposed to the test substance at nominal concentrations of 3.125, 6.25, 12.5, 25, 50 and 100 mg/L, corresponding to the geometric mean measured concentrations of 2.14, 4.46, 8.38, 16.6, 35.4 and 67.9 mg/L for 72 h. Three replicates were tested for each test substance concentration and six replicates for the control. The environmental conditions were within the acceptable limits. 

The concentrations of the test substance were analytically verified by GC-MS at the start and the end of the exposure of all test concentrations and the control. The recovery rate of the test substance at the start of the exposure was in the range of 92 to 108 % of the nominal values at all tested concentration levels. The recovery rate at the end of the exposure was in the range of 45 to 49 % of the nominal values. All effect values were based on the geometric mean measured concentrations of the test substance.

All validity criteria of the test guideline were fulfilled (Increase of cell growth in the control cultures, mean coefficients of variation for specific growth rates and coefficient of variation of average specific growth rates).

In this study, the test substance was found to inhibit the growth of the freshwater green algae Pseudokirchneriella subcapitata with the following effect values (geometric mean measured test item concentrations): The EC50 value with 95 % CI for inhibition of growth rate (ErC50) after 72 hours was > 67.9 mg/L. The EC10-value with 95 % CI for inhibition of growth rate (ErC10) after 72 hours was 27.5 (24.9 - 30.3) mg/L. The NOEC-value for inhibition of growth rate after 72 hours was 16.6 mg/L.