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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
6 Mar - 28 Apr 2017
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: main test not completed
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Appearance: off-white waxy solid
- Source and lot/batch No.of test material: 629582
- Expiration date of the lot/batch: 9 Dec 2017
- Purity: 98.3%
- Purity test date: 14 Jun 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Substance is hydrolytically active
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 2.0 mL
- Sampling method: Samples were taken at the start of the test, after 24 hours, and after 72 hours.
- Sample storage conditions before analysis: Samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: All glassware used was silanized to minimise adsorption. Preparation of test solutions started with a loading rate of 100 mg/L applying a two-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. This resulted in a hazy mixture. The aqueous Saturated Solution (SS) was collected by means of filtration through a 0.45 μm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- Controls: no
- Evidence of undissolved material: All test solutions were clear and colorless at the end of the preparation.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: NIVA CHL1
- Source: in-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1) 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 and light intensity of 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. Growth (M1) medium (Nederlandse Praktijk Richtlijn no. 6505) was formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition.
NaNO3, 500 mg/l
K2HPO4∙3H2O, 52 mg/l
MgSO4∙7H2O, 75 mg/l
Na2CO3∙10H2O, 54 mg/l
C6H8O7∙H2O, 6 mg/l
NH4NO3, 330 mg/l
CaCl2∙2H2O, 35 mg/l
C6H5FeO7∙xH2O, 6 mg/l
H3BO3, 2.9 mg/l
MnCl2∙4H2O, 1.81 mg/l
ZnCl2, 0.11 mg/l
CuSO4∙5H2O, 0.08 mg/l
(NH4)6Mo7O24∙4H2O, 0.018 mg/l
- Acclimation period: Four days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E4 cells/mL. The pre-culture was maintained under the same conditions as used in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22 - 23 °C
pH:
7.2 - 8.1
Nominal and measured concentrations:
Nominal: 1.0, 10, 100% saturated solution
Measured: 0.0067, 0.39, and 0.16 μg/L (at start of test, extrapolated from calibration curve)
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, silanized
- Type: open
- Fill volume: 50 mL
- Aeration: continuous shaking
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 70.5E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, OECD medium (M2) prepared in Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 84 to 89 μE.m-2.s-1. Test vessels were placed randomly in the incubator, and repositioned daily.

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 at the end of the test by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length = 10 mm. Algal medium was used as blank and the extra replicates as background for the treated solutions.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2CrO7)
Key result
Duration:
72 h
Dose descriptor:
EC50
Remarks on result:
not measured/tested
Remarks:
Information became available showing that the test item is subject to rapid hydrolysis
Details on results:
The mean cell densities measured during the combined limit/range-finding test are presented in Table 1. Table 2 presents the percentages growth rate inhibition per concentration. Effects on algal growth were minimal at 1.0% SS, while algal growth was near to completely inhibited at 10 and 100% SS. Therefore, the expected EC50 for growth rate was between test groups representing 1.0 and 10% SS. However, the 72-hour EC50 for Pseudokirchneriella subcapitata exposed to MTDID 15670 was not determined. The project was stopped in consultation with the sponsor. Information became available showing that the test item is subject to rapid hydrolysis. The sponsor decided to perform additional testing on the hydrolysis products instead.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.99 mg/L (growth rate). The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L.
- Other: The reference substance test was conducted within three months of the testing of MTDID 15670.

Table 1. Mean Cell Densities (x1E+04 cells/mL) During the Range-Finding Test

 Time (h)  Control  1.0% SS  10% SS  100% SS
 0  1.0  1.0  1.0  1.0
 72  70.5  57.9  2.1  1.6

Table 2. Percentage Inhibition of Growth Rate During the Range-Finding Test

 Test Group  Mean  Std. Dev.  n  % Inhibition
 Control  1.412  0.0861  3  
 1.0% SS  1.352  0.0406  3  4.3
 10% SS  0.238  0.1063  3  83.1
 100% SS  0.131  0.1273  3  90.8
Validity criteria fulfilled:
not applicable
Conclusions:
The 72-hour NOEC and EC50 for Pseudokirchneriella subcapitata exposed to MTDID 15670 in a range-finding test according to OECD 201 were not determined. The project was stopped before the range-finding tests were completed. Information became available showing that the test item is subject to rapid hydrolysis.
Executive summary:

A range-finding test preliminary to a full study as per OECD 201 was done. The 72-hour NOEC and EC50 for Pseudokirchneriella subcapitata exposed to MTDID 15670 were not determined. The project was stopped before the range-finding test series for all species was completed. Information became available showing that the test item is subject to rapid hydrolysis. Because the test was not completed, the reliability of the results could not be assigned.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
02-March-2018 to 16-March-2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
Dodecyl methyl sulfide is used within this study as an analogous chemical to dodecyl ethyl sulfide, which is a hydrolysis product of MTDID 15670. Dodecyl ethyl sulfide is not commercially available.
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sigma-Aldrich, Batch Number MKBW6054V
- Physical state: colorless, liquid
- Quality Release Date: 12JAN2016
- Purity: 97.8%
- Storage condition of test material: room temperature
Analytical monitoring:
yes
Details on sampling:
- Concentrations: negative control and solutions containing 1.0, 10, and 100% of the saturated solution (SS) prepared at a loading rate of 100 mg/L (limit test).
- Volume: Control: 3 mL for the regular samples and 1.5 mL for quality control (QC) samples
- Sampling method: Duplicate samples for analysis, and duplicate reserve samples, were taken from the test vessels. Two extra samples were taken from the limit concentration (100% SS) for analytical QC, and two reserve QC samples were also taken. Samples were taken at test initiation (T = 0 h), T= 24 h, and test completion (T= 96 h).
- Sample storage conditions before analysis: Samples were transferred to the analytical laboratory at the test facility on the day of sampling for extraction then stored in a freezer (≤ -15°C) until analyses.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The batch of dodecyl methyl sulfide tested was a colorless liquid which was not completely soluble in test medium at the initially prepared loading rate. Preparation of test solutions started with a loading rate of 100 mg/L applying a two-day period of gentle magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period o fone day. 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.
- Controls: Blank only
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Test solutions were clear and colorless at the end of the preparation procedure.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: NIVA CHL1
- Source: in-house laboratory culture.
- Age of inoculum (at test initiation): Three days
- Method of cultivation: Stock cultures were started by inoculating growth (M1) medium with algal cells from a pure culture on agar. The suspensions were continuously aerated in a climate room at a temperature of 21-24°C and light intensity of 60 to 120 μE/m²/s when measured in the photosynthetically effective wavelength range (400-700 nm). Growth (M1) medium (Nederlandse Praktijk Richtlijn no. 6505) was formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition.
NaNO3, 500 mg/l
K2HPO4∙3H2O, 52 mg/l
MgSO4∙7H2O, 75 mg/l
Na2CO3∙10H2O, 54 mg/l
C6H8O7∙H2O, 6 mg/l
NH4NO3, 330 mg/l
CaCl2∙2H2O, 35 mg/l
C6H5FeO7∙xH2O, 6 mg/l
H3BO3, 2.9 mg/l
MnCl2∙4H2O, 1.81 mg/l
ZnCl2, 0.11 mg/l
CuSO4∙5H2O, 0.08 mg/l
(NH4)6Mo7O24∙4H2O, 0.018 mg/l
- Acclimation period: Three days before the start of the test, fresh M2 medium was inoculated at adensity of 1E4 cells/mL and was kept under the same conditions used in the test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
22 °C to 23 °C
pH:
8.1
Nominal and measured concentrations:
Nominal: 100 mg/L loading level (100% SS), 10% SS, 1% SS, Control (Blank)
Measured: Only the 100 mg/L (100% SS) loading level and the blank were analyzed. A quantifiable response was not measured at any concentration, but estimated concentrations were determined for some samples by extrapolation of the standard curve (See Table 1 for analysis results). Based on these estimated values, a maximum soluble concentration was conservatively estimated to be 0.03 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass vessels
- Fill volume: 50 mL
- Aeration: continuous shaking
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 245E+04 cells/mL
- No. of vessels per concentration: Six replicates for limit concentration (100%SS) three replicates for 1.0% and 10% SS. One extra replicate for each test group for sampling purposes after 24 h of exposure. One or two replicates of each test concentration without algae.
- No. of vessels per control: Six replicates
GROWTH MEDIUM
- Standard medium used: Yes, OECD medium (M2)
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Growth medium
- Culture medium different from test medium: No
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuous
- Light intensity and quality: TLD lamps with a light intensity within the range of 84 to 85 μE/(m²∙s).
Test vessels were placed randomly in the incubator, and repositioned daily.
EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cells were counted using a microscope and a counting chamber to determine inoculum density. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength = 10 mm) against an algal medium blank and the extra replicates for the treated solutions.
- Recording times: cell densities were recorded at 24-hour intervals in the control and limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: Ten
- Justification for using less concentrations than requested by guideline: Limit test
RANGE-FINDING STUDY
- Combined limt/range-finding study
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2CrO7)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
based on loading level of 100 mg/L, which is above the water solubility of dodecyl methyl sulfide.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
based on loading level of 100 mg/L, which is above the water solubility of dodecyl methyl sulfide.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.03 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
0.03 mg/L was estimated to be the maximum soluble concentration in the medium, and was outside the range of the standard curve.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.03 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
0.03 mg/L was estimated to be the maximum soluble concentration in the medium, and was outside the range of the standard curve.
Details on results:
Under the conditions of the study no biologically significant inhibition of growth rate was observed at a loading rate of 100 mg/L. 14% inhibition of yield was observed at a loading rate of 100 mg/L.
- Exponential growth in the control: yes.
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: yes
Results with reference substance (positive control):
Results with reference substance valid? yes
- EC50: 1.6 mg/L (growth rate). Historical range for the reference substance at the contract lab lies between 0.82 and 2.3 mg/L
- Other: The reference substance test was conducted within one month of testing dodecyl methyl sulfide.

Table 2, Growth Rate and Percentage Inhibition for the Total Test Period

Dodecyl Methyl Sulfide* Mean Std. Dev. n % Inhibition
Control 1.833 0.0194 6  
1.0 1.827 0.0162 3 0.3
10.0 1.831 0.0307 3 0.1
100 1.783 0.0467 3 2.7**

* Percentage of saturated solution prepared at 100 mg/L loading rate

** Effect was statistically, but not biologically significant

Validity criteria fulfilled:
yes
Remarks:
Control cell density increased by a factor of > 16 (i.e. 245); mean CV for section-by-section specific growth rate in the controls was < 35% (i.e. 14%); CV of average specific growth rates in the whole test period for controls was < 7% (i.e. 1.1%).
Conclusions:
The 72-hour EC10 and EC50 (growth rate) of dodecyl methyl sulfide to Pseudokirchneriella subcapitata exceeded a loading rate of 100 mg/L, which exceeded the maximum soluble concentration for dodecyl methyl sulfide in test medium. The test was conducted according to OECD TG 201.
Executive summary:

The 72-hour EC10 and EC50 of dodecyl methyl sulfide to Pseudokirchneriella subcapitata were examined in a static test conducted according to OECD TG 201. Dodecyl methyl sulfide is a surrogate for dodecyl ethyl sulfide (not commercially available), a hydrolysis product of MTDID 15670. Test solutions were prepared as the control (0 mg/L), and a 100 mg/L loading level (water soluble fraction). Due to the very low solubility of dodecyl methyl sulfide, analytical measurement failed to quantify the compound in any samples. Extrapolation of the calibration curve allowed estimation of the concentration in some samples (0 h, 24 h), leading to an estimated maximum soluble concentration of 0.03 mg/L in the test medium. The 72-hour EC10 and EC50 for growth rate were > 100 mg/L (nominal loading concentration), and therefore greater than the water solubility of dodecyl methyl sulfide in the test medium. The test was conducted according to internationally accepted test guidelines and was GLP compliant. It is reliable with restrictions and suitable for Risk Assessment, Classification and Labeling, and PBT Analysis in a weight of evidence (WOE) approach.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
26 Feb - 31 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- State of substance: clear colourless liquid
- Source and lot/batch No.of test material: Sigma-Aldrich Chemie GmbH, Steinheim, Germany, batch:
STBD0697V
- Purity: 99.8%, mixture of cis and trans

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 mg/L, 0.1 mg/L and 100 mg/L
- Sampling method: Samples were taken at the start of the test, at 24 hours, and at 72 hours.
- Sample storage conditions before analysis: Samples were transferred to the analytical laboratory at the Test Facility on the day of sampling for extraction and then stored in a freezer (≤-15°C) until analyses.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L (weighed amount of 99.4 mg in 1 liter of medium) applying 10 minutes of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Controls: Test medium without test item or other additives
- Evidence of undissolved material: All test solutions were clear and colorless at the end of the preparation procedure.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: NIVA CHL1
- Source: in-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1) 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 and light intensity of 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. Growth (M1) medium (Nederlandse Praktijk Richtlijn no. 6505) was formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition.
NaNO3, 500 mg/l
K2HPO4∙3H2O, 52 mg/l
MgSO4∙7H2O, 75 mg/l
Na2CO3∙10H2O, 54 mg/l
C6H8O7∙H2O, 6 mg/l
NH4NO3, 330 mg/l
CaCl2∙2H2O, 35 mg/l
C6H5FeO7∙xH2O, 6 mg/l
H3BO3, 2.9 mg/l
MnCl2∙4H2O, 1.81 mg/l
ZnCl2, 0.11 mg/l
CuSO4∙5H2O, 0.08 mg/l
(NH4)6Mo7O24∙4H2O, 0.018 mg/l
- Acclimation period: Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E4 cells/mL. The pre-culture was maintained under the same conditions as used in the test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
23 °C
pH:
8.0 - 8.4
Nominal and measured concentrations:
Nominal: 100 mg/L
Measured: 17 mg/L (time weighted average)
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass
- Fill volume: 50 mL
- Aeration: continuousl shaking
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 252.1E+04 cells/mL
- No. of vessels per concentration (replicates): 6 replicates of limit concentration, 3 replicates of other test concentrations
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD medium (M2) prepared in Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 86 to 87 μE.m-2.s-1. Test vessels were placed randomly in the incubator, and repositioned daily.

EFFECT PARAMETERS MEASURED :
- 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 at the end of the test by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length = 10 mm. Algal medium was used as blank and the extra replicates as background for the treated solutions.

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1.0, 10, and 100 mg/L, contained within limit test
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2CrO7)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 17 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No inhibition of growth rate at highest concentration tested.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
17 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No inhibition of growth rate at highest concentration tested.
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.86 mg/L (growth rate). The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L.
- Other: The reference substance test was conducted within two months of the testing of MTDID 15670.

Table 1. Growth Rate And Percentage Inhibition For The Total Test Period

 Crotononitrile nominal conc. (mg/L)  Mean  Std. Dev.  n  %Inhibition
 Control  1.843  0.0119  6  
 0.10  1.835  0.0052  3  0.45
 1.0  1.848  0.0090  3  -0.27
 10  1.849  0.0041  3  -0.32
 100 (TWA: 17)  1.850  0.0077  6  -0.38
Validity criteria fulfilled:
yes
Remarks:
Control cell density increased by a factor of > 16 (252); The mean CV for section-by-section specific growth rate in the controls was < 35% (12%); the CV of average specific growth rates in the whole test period for controls was < 7% (0.65%).
Conclusions:
The 72-hour EC50 (growth rate) of crotononitrile, a hydrolysis product of MTDID 15670, to Pseudokirchneriella subcapitata was > 17 mg/L (OECD 201).
Executive summary:

The 72-hour EC50 (growth rate) of crotononitrile, a hydrolysis product of MTDID 15670, to Pseudokirchneriella subcapitata was examined in a range-finding/limit static test conducted according to OECD 201. Test solutions were prepared as the control (0 mg/L), 0.1 mg/L, 1 mg/L, 10 mg/L, and 100 mg/L. At the end of the test, no inhibition of growth rate was observed at the highest test level, which had a time weighted average (TWA) concentration of 17 mg/L. The EC50 is therefore > 17 mg/L. The study was well-documented, followed an international standard method, and was GLP compliant; therefore, the test is considered reliable without restriction.

7

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
Singular 2 mL samples for possible analysis were taken from all test concentrations and the control (centre of test vessels) at t=0 h, t=24 h and t=48 h. Samples were stored in the freezer (≤ -15°C) until analysis.
On the day of analysis, the samples were defrosted at room temperature. The test samples at 100 mg/L were diluted 100-fold with M2-medium. Finally, all (pre-diluted) test samples were diluted 100-fold in 50/50 (v/v) methanol/water to obtain concentrations within the calibration range.
Vehicle:
no
Details on test solutions:
PREPARATION OF TEST SOLUTIONS
The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film of the test substance on the water surface). The batch of Potassium tetrafluoroborate tested was a white powder with a purity of 99.1% (w/w) and completely soluble in test medium at the concentrations tested. Preparation of test solutions started with the highest test concentration of 100 mg/L. No special treatment other than careful mixing was necessary to completely dissolve the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions in test medium. The final test solutions were all clear and colourless. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 1E+04 cells/mL.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM:
- Strain: NIVA CHL 1
- Source: 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. Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Initial cell density: 1E+04 cells/mL as determined using a microscope and a counting chamber

ACCLIMATION
- Acclimation period: 3 days.
- Culturing media and conditions: The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Test temperature:
22.1-23.1°C
pH:
7.6 - 8.2
Nominal and measured concentrations:
- Nominal: 0.10, 1.0, 10 and 100 mg/L.
- Measured: Analysis of the samples taken from 100 mg/L showed a measured concentration of 116 mg/L at the start of exposure. The concentration remained stable during the 72-hour test period (96-101% of initial).
Details on test conditions:
TEST SYSTEM:
- Test vessel: 100 ml, all-glass, containing 50 mL of test solution
- Type: closed
- Initial cells density: 1E+04 cells/mL
- No. of vessels (replicates): 6 replicates of the control and 100 mg/L, 3 replicates of 0.10, 1.0 and 10 mg/L, 1 extra replicate of each test group for sampling purposes after 24 hours, 1 or 2 replicates of each test concentration without algae.

GROWTH MEDIUM:
- Standard medium used: yes (M2)

OTHER TEST CONDITIONS:
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuous illumination
- Light intensity and quality: 30 Watt "Cool White" TLD lamps with a light intensity within the range of 78 to 90 µE.m-2.s-1

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
No significant reduction of growth rate or inhibition of yield was recorded at any of the concentrations of Potassium tetrafluoroborate tested. All results were based on nominal concentrations despite the fact that analytical results showed a slightly higher concentration than nominal. This was justified due to the fact that procedural recovery samples at 100 mg/L showed recoveries in the same range of nominal. No EC50-values could be calculated because the test substance proved to be non-toxic (EC50 > maximum concentration tested).
Results with reference substance (positive control):
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.5 mg/L with a 95% confidence interval ranging from 1.1 to 2.1 mgL. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/L. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
Statistical analysis of the data was not needed as the effects recorded were not significant (<10%).
Validity criteria fulfilled:
yes
Conclusions:
Pseudokirchnerella subcapitata exposed to 0.10, 1.0, 10 and 100 mg/L potassium tetrafluoroborate under specific conditions for 72 hours displayed no significant reduction of growth rate or inhibition of yield. The 72-h ErC50 and EyC50 were >100 mg/L and the NOErC was 100 mg/L (based on nominal concentrations).
Executive summary:

In order to test the toxicity of potassium tetrafluoroborate to freshwater alga, a GLP-compliant study was performed according to OECD TG 201. In this study, Pseudokirchnerella subcapitata were exposed to potassium tetrafluoroborate in a combined limit/range-finding test for 72 hours under static conditions. Alga, at an initial cell density of 1E+04 cells/mL, were exposed to 100 mg/L and a blank control in the limit test (6 replicates per test group) and to concentrations of 0.10, 1.0 and 10 mg/L in the range-finding test (3 replicates per test group). Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. Analysis of the samples taken from 100 mg/L showed a measured concentration of 116 mg/L at the start of exposure. The concentration remained stable during the 72-hour test period (96-101% of initial). All results were based on nominal concentrations despite the fact that analytical results showed a slightly higher concentration than nominal. This was justified due to the fact that procedural recovery samples at 100 mg/L showed recoveries in the same range, i.e. 115-116% of nominal. No significant reduction of growth rate or inhibition of yield was recorded at any of the concentrations of potassium tetrafluoroborate tested. No EC50 could be calculated because the test substance proved to be non-toxic (EC50 > maximum concentration tested). Therefore, the 72-h ErC50 (growth rate) and EyC50 (biomass) values were determined at >100 mg/L (based on nominal concentrations); the NOEC value was determined at 100 mg/L for both parameters.

Description of key information

MTDID 15670 hydrolyzes rapidly (T1/2 < 12 hrs) in water. Results from toxicity tests (OECD TG 201) with the hydrolysis products of MTDID 15670 (or a surrogate chemical) all indicate that with Raphidocelis subcapitata the 72-hour EC50 and 72-hour NOEC/EC10 are > 100 mg/L.

Key value for chemical safety assessment

Additional information

A preliminary toxicity to algae study (OECD 201) with MTDID 15670 for Raphidocelis subcapitata was initiated, but stopped after consultation with the sponsor, after encountering phase separation (precipitation) during the range-finding test. Some inhibition of growth rate was observed in the range-finding test, however the NOEC and EC50 were not determined. Subsequently, information became available showing that MTDID 15670 is subject to rapid hydrolysis. Because the test was not completed, the reliability of the results could not be assigned. Analysis after hydrolysis revealed three products; two chemicals with moderate water solubility, crotononitrile (CAS# 4786-20-3) and tetrafluoroborate anion (tetrafluoroboric acid, CAS# 16872-11-0), along with the poorly soluble chemical, dodecyl ethyl sulfide (No CAS#). Short-term toxicity testing results for these hydrolysis products follow.

The 72-hour NOEC and EC50 of crotononitrile, to Raphidocelis subcapitata was examined in a rangefinding/static limit test conducted according to OECD TG 201. Test solutions were prepared as the control (0 mg/L), 0.1 mg/L, 1 mg/L, 10 mg/L, and at 100 mg/L. No inhibition of growth rate was seen at the 100 mg/L loading rate. The measured concentration at the highest loading rate was 17 mg/L. The study was well-documented, followed an international standard method, and was GLP compliant; therefore, the test is considered reliable without restriction.

The 72-hour EC10 and EC50 of dodecyl methyl sulfide (CAS# 3698-89-3) to Raphidocelis subcapitata were examined in a static, limit test conducted according to OECD TG 201. Dodecyl methyl sulfide is a surrogate for dodecyl ethyl sulfide (not commercially available). Test solutions were prepared as the control (0 mg/L) and as a water soluble fraction at a loading rate of 100 mg/L. The 72-hour EC10 and EC50 (growth rate) exceeded the loading rate of 100 mg/L. Due to the very low solubility of dodecyl methyl sulfide, analytical measurement failed to quantify the compound in any samples. Extrapolation of the calibration curve allowed estimation of the concentration in some samples (0 h, 24 h), leading to an estimated maximum soluble concentration of 0.03 mg/L in the test medium.. The study was well-documented, followed an international standard method, and was GLP compliant. The study is considered reliable with restrictions. The results from this study are considered suitable for Risk Assessment, Classification and Labeling, and PBT Analysis.

The toxicity of tetrafluoroboric acid, to Rapidocelis subcapitata was examined as potassium tetrafluoroborate in a rangefinding/limit test conducted according to OECD201. Test solutions were prepared as the control (0 mg/L), 0.1 mg/L, 1 mg/L, 10 mg/L, and at 100 mg/L. No inhibition of growth rate was seen at the 100 mg/L loading rate. No significant reduction of growth rate or inhibition of yield was recorded at any of the concentrations. Analysis of the 100 mg/L samples showed a measured concentration of 116 mg/L at the start of exposure. The study followed an international standard method, and was GLP compliant; therefore, the test is considered reliable without restriction.