3-methyl-1-isobutylbutyl acetate

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:

03 June 2019 to 12 July 3019

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:

23 March 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals, OECD series on testing and assessment number 23

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples for possible analysis were taken from all test concentrations and the control.
- Sampling method: Frequency: at t = 0, 24 and 72 h; volume: 6.0 mL from the approximate centre of the test vessels.
- Sample storage conditions before analysis: During the final test, 100 μL acetone was added to the samples before storage in a freezer at the start of the test and at t = 24 h, in accordance with the analytical method. Inadvertently, acetone was not added to samples taken at the end of the test). Storage Samples were stored in a freezer (≤-15 °C) until analysis.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 10 % of the SS but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period. Additionally, reserve samples of 6.0 mL were taken from all test solutions for possible analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The batch tested was not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition. All glassware used during the preparation of test solutions was closed with minimal headspace to minimise vaporisation of test material.
Preparation of test solutions started with a loading rate of 100 mg/L applying a one-day period of magnetic stirring to ensure maximum dissolution of the test material in medium. The obtained mixture was allowed to settle for a period of 30 minutes. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colourless at the end of the preparation procedure. Furthermore, tyndall effect was checked with the use of a laser pen, no tyndall effect was observed.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Raphidocelis subcapitata, strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21 - 24 °C. The light intensity was 60 to 120 μE/m²/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium was M1 formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition (all concentrations in mg/L): NaNO3 500, K2HPO4 39.5, MgSO4.7H2O 75, Na2CO3 20, C6H8O7.H2O 6, NH4NO3 330, CaCl2.2H2O 35, C6H5FeO7.xH2O 6, H3BO3 2.9, MnCl2.4H2O 1.81, ZnCl2 0.11, CuSO4.5H2O 0.08 and (NH4)6Mo7O24.4H2O 0.018.

PRE-CULTURE
4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL.
- Culturing media and conditions (same as test or not): The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use. The pre-culture medium was adjusted M2, formulated using Milli-RO water and with the following 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 64 μg/L, Na2EDTA.2H2O 100 μg/L, H3BO3 185 μg/L, MnCl2.4H2O 415 μg/L, ZnCl2 3 μg/L, CoCl2.6H2O 1.5 μg/L, CuCl2.2H2O 0.01 μg/L, Na2MoO4.2H2O 7 μg/L, NaHCO3 300 mg/L and HEPES buffer 6 mmol/L. The pH was 7.1 ± 0.3.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 - 23 °C

pH:

7.5 - 7.9

Nominal and measured concentrations:

- Nominal concentrations: Solutions containing 1.0, 3.2, 10, 32 and 100 % of the SS, prepared at a loading rate of 100 mg/L.
- Time Weighted Average (TWA) concentrations were 0, 0.054, 0.22, 0.59, 0.53, 1.5 and 8.8 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 40 mL, glass vials
- Type: Closed (airtight)
- Material, size, headspace, fill volume: no headspace to prevent any loss of the test material due to volatilization
- Aeration: No; during incubation the algal cells were kept in suspension by continuous shaking
- Initial cell density: An initial cell density of 1 x 10^4 cells/mL.
- Control end cell density: 1.24 x 10^6
- No. of vessels per concentration (replicates): 3 replicates of each test concentration, 1 extra replicate of each test group for sampling purposes after 24 hours of exposure, 1 or 2 replicates of each test concentration without algae
- No. of vessels per control (replicates): 6 replicates of the control
- Preparation of test solutions: After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate, providing a cell density of 10^4 cells/mL. Any residual volumes were discarded.

GROWTH MEDIUM
- Standard medium used: Yes; Test Medium Adjusted M2 same as used for the pre-culture

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Medium was formulated using Milli-RO water
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test, for all test concentrations and the control. The temperature of the medium was measured continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: TLD-lamps with a light intensity within the range of 74 to 78 μE.m^-2.s^-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Other: At the end of the final test microscopic observations were performed on the 32 % of the SS test solution and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Yes
- Test concentrations: Control and a Saturated Solution (SS) prepared at a loading rate of 100 mg/L (6 replicates) and 1.0 and 10 % of the SS prepared at a loading rate of 100 mg/L (3 replicates)
- Results used to determine the conditions for the definitive study: Yes. No inhibition of algal growth rate or yield was found at 1.0 % of the SS. Dose-related inhibition of algal growth rate and yield was found at 10 and 100 % of the SS at the end of the test. An inhibition of 83 and 99 % was observed at the highest test concentration, respectively. Based on these results, samples taken from solutions containing 1.0 and 100 % of the SS prepared at a loading rate of 100 mg/L were analysed. The measured concentrations at the start of the test were 0.15 and 12 mg/L, respectively. During the exposure period, the concentration analysed at 1.0 % of the SS remained stable (i.e. was at 100 % of initial at the end of the test) while the concentration analysed at 100 % of the SS decreased to 67 % of initial at the end of the test.

INTERPRETATION
- Acceptability of the Test
1. In the control, cell density increased by an average factor of at least 16 within the exposure period.
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35 %.
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

- Calibration Curve
Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.

- Comparison of Average Growth Rates
The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments:
µi-j = (lnXj - lnXi) / (tj - ti) Day^-1
Where:
μi-j = the average specific growth rate from time i to j
Xi = the biomass at time i
Xj = the biomass at time j

The average growth rate at each test material concentration is then compared with the control value and the percentage inhibition in growth rate is calculated:
%Ir = ((µC - µT) / µC) x 100
Where:
%Ir = percent inhibition in average specific growth rate
μC = mean value for average specific growth rate in the control group
μT = average specific growth rate for the treatment replicate

- Yield
The percent inhibition in yield is calculated for each treatment replicate as follows:
%Iy = ((YC - YT) / YC) x 100
Where:
%Iy = percent inhibition of yield
YC = mean value for yield in the control group
YT = value for yield for the treatment replicate

- Determination of the Average Exposure Concentrations
The average exposure concentrations were calculated as: [(24 x √(Ct = 0 x Ct = 24)) + (48 x √(Ct = 24 x Ct = 72))] / 72 being the Time Weighted Average (TWA) of the concentrations measured in the samples taken at the start (Ct = 0), after 24 hours (Ct = 24) and the end of the test (Ct = 72).

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 (potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 8.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CL 1.2 - 1.6 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.59 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.:

0.22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

INHIBITION OF GROWTH RATE AND INHIBITION OF YIELD
Table 1 shows group mean growth rates and the percentages of growth rate inhibition (total test period). The group mean yields and the percentages of yield inhibition are summarised in Table 2.
Inhibition of growth rates and yield increased with increasing concentration of the test material from 0.59 mg/L upwards resulting in 45 % growth rate and 88 % yield inhibition at the highest TWA concentration of 8.8 mg/L. Statistically significant inhibition of growth rates was found at concentrations of 1.5 mg/L and higher. Statistically significant inhibition of yield was found at concentrations of 0.59 mg/L and higher.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 32 mg/L when compared to the control.
In conclusion, under the conditions of the present study with Raphidocelis subcapitata, the test material inhibited growth rate of this freshwater algae species significantly at TWA concentrations of 1.5 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range of concentrations tested, i.e. exceeded a TWA concentration of 8.8 mg/L.

Results with reference substance (positive control):

The reference test was carried out to check the sensitivity of the test system used by the test facility to potassium dichromate. Algae were exposed for a period of 72 hours to nominal K2Cr2O7 concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 10^4 cells/mL.
Potassium dichromate inhibited growth rate at nominal concentrations of 0.18 mg/L and higher. The 72h-EC50 for growth rate inhibition (ERC50) was 1.11 mg/L with a 95 % confidence interval ranging from 1.10 to 1.13 mg/L. The 72h-ERC50 was within the expected range of 0.92 and 1.46 mg/L and the historical range of 0.86 and 2.3 mg/L based on reference tests performed at the test facility during the last ten years.
The study met the acceptability criteria prescribed by the study plan and was considered valid.

Reported statistics and error estimates:

DETERMINATION OF THE NOEC AND CALCULATION OF THE EC50
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Step-down Jonckheere-Terpstra Test Procedure, α = 0.05, one-sided, smaller) or inhibition of yield (Williams Multiple Sequential t-test Procedure, α = 0.05, one-sided, smaller).
Calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test material.
The ErC50-value could not be determined because the observed effect was below 50 %. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Growth Rate and Percentage Inhibition for the Total Test Period

TWA concentration (mg/L)	Mean	Std. Dev.	n	% Inhibition
Control	1.606	0.0206	6
0.054	1.611	0.0159	3	-0.31
0.22	1.621	0.0111	3	-0.93
0.59	1.571	0.0227	3	2.2
1.5	1.337	0.0951	3	17*
8.8	0.878	0.1920	3	45*

* Effect was statistically significant

 

Table 2: Yield and Percentage Inhibition for the Total Test Period

TWA concentration (mg/L)	Mean	Std. Dev.	n	% Inhibition
Control	123.0	7.80	6
0.054	124.7	5.91	3	-1.4
0.22	128.5	4.30	3	-4.5
0.59	110.6	7.51	3	10*
1.5	55.7	14.83	3	55*
8.8	14.5	8.14	3	88*

* Effect was statistically significant

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test material inhibited growth rate significantly at TWA concentrations of 1.5 mg/L and higher. The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range of concentrations tested, i.e. exceeded a TWA concentration of 8.8 mg/L. The 72h-EC50 for yield inhibition (EYC50) was 1.4 mg/L with a 95 % confidence interval ranging from 1.2 to 1.6 mg/L. The 72h-NOEC for growth rate inhibition was 0.59 mg/L. The 72h-NOEC for yield inhibition was 0.22 mg/L

Executive summary:

A toxicity test with Raphidocelis subcapitata was carried out in accordance with the standardised guidelines OECD 201 and OECD series on testing and assessment number 23 under GLP conditions.

The batch of material tested was a clear colourless liquid and not completely soluble in test medium at the loading rate initially prepared. All glassware used during the preparation of test solutions was airtight closed with minimal headspace to minimise vaporisation of test material.

A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to solutions containing 1.0, 3.2, 10, 32 and 100 % of the SS prepared at a loading rate of 100 mg/L. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. No test material was detected in the control throughout the test. The measured concentrations at the start of the test were 0.10, 0.40, 1.1, 3.6 and 13 mg/L, respectively. During the exposure period, the concentrations decreased to 8 - 34 % of initial at the end of the test. Based on these results, the average exposure concentrations were calculated and used to express effect parameters. The Time Weighted Average (TWA) concentrations were 0, 0.054, 0.22, 0.59, 0.53, 1.5 and 8.8 mg/L.

Inhibition of growth rates and yield increased with increasing concentration of test material from 0.59 mg/L upwards resulting in 45 % growth rate and 88 % yield inhibition at the highest TWA concentration of 8.8 mg/L. Statistically significant inhibition of growth rates was found at concentrations of 1.5 mg/L and higher. Statistically significant inhibition of yield was found at concentrations of 0.59 mg/L and higher.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the test material inhibited growth rate significantly at TWA concentrations of 1.5 mg/L and higher. The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range of concentrations tested, i.e. exceeded a TWA concentration of 8.8 mg/L. The 72h-EC50 for yield inhibition (EYC50) was 1.4 mg/L with a 95 % confidence interval ranging from 1.2 to 1.6 mg/L. The 72h-NOEC for growth rate inhibition was 0.59 mg/L. The 72h-NOEC for yield inhibition was 0.22 mg/L

Description of key information

Under the conditions of this study, the test material inhibited growth rate significantly at TWA concentrations of 1.5 mg/L and higher. The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range of concentrations tested, i.e. exceeded a TWA concentration of 8.8 mg/L. The 72h-EC50 for yield inhibition (EYC50) was 1.4 mg/L with a 95 % confidence interval ranging from 1.2 to 1.6 mg/L. The 72h-NOEC for growth rate inhibition was 0.59 mg/L. The 72h-NOEC for yield inhibition was 0.22 mg/L

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.59 mg/L

Additional information

A toxicity test with Raphidocelis subcapitata was carried out in accordance with the standardised guidelines OECD 201 and OECD series on testing and assessment number 23 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The batch of material tested was a clear colourless liquid and not completely soluble in test medium at the loading rate initially prepared. All glassware used during the preparation of test solutions was airtight closed with minimal headspace to minimise vaporisation of test material.

A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to solutions containing 1.0, 3.2, 10, 32 and 100 % of the SS prepared at a loading rate of 100 mg/L. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. No test material was detected in the control throughout the test. The measured concentrations at the start of the test were 0.10, 0.40, 1.1, 3.6 and 13 mg/L, respectively. During the exposure period, the concentrations decreased to 8 - 34 % of initial at the end of the test. Based on these results, the average exposure concentrations were calculated and used to express effect parameters. The Time Weighted Average (TWA) concentrations were 0, 0.054, 0.22, 0.59, 0.53, 1.5 and 8.8 mg/L.

Inhibition of growth rates and yield increased with increasing concentration of test material from 0.59 mg/L upwards resulting in 45 % growth rate and 88 % yield inhibition at the highest TWA concentration of 8.8 mg/L. Statistically significant inhibition of growth rates was found at concentrations of 1.5 mg/L and higher. Statistically significant inhibition of yield was found at concentrations of 0.59 mg/L and higher.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the test material inhibited growth rate significantly at TWA concentrations of 1.5 mg/L and higher. The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range of concentrations tested, i.e. exceeded a TWA concentration of 8.8 mg/L. The 72h-EC50 for yield inhibition (EYC50) was 1.4 mg/L with a 95 % confidence interval ranging from 1.2 to 1.6 mg/L. The 72h-NOEC for growth rate inhibition was 0.59 mg/L. The 72h-NOEC for yield inhibition was 0.22 mg/L