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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9-April-2018 to 12-April-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
28-July-2011
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
N-methyldiallylamine
EC Number:
219-354-4
EC Name:
N-methyldiallylamine
Cas Number:
2424-01-3
Molecular formula:
C7H13N
IUPAC Name:
methylbis(prop-2-en-1-yl)amine
Specific details on test material used for the study:
- Source and lot/batch No.of test material: Lot 20160912
- Purity: 99.2%
- Expiration date of the lot/batch: 1-August-2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: test substance stable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: negative control (0 mg/L), 1.0, 3.2, 10, 32, and 100 mg/L.
- Volume: Control: 3.6 mL for the regular samples and 1.8 mL for quality control (QC) samples
- Sampling method: Duplicate samples for analysis, and reserve samples, were taken from each concentration before transfer to the replicate test vessels for the initial (T = 0 hours) samples. Two extra samples of 1.8 mL were taken from the 10 mg/L test group for analytical QC, and reserve QC samples were also taken. At test completion (T = 72 hours) test solutions were not pooled, due to the volatility of MTDID 49220, and duplicate samples were taken from at least one individual replicate.
- 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.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The batch of MTDID 49220 tested was a clear yellow liquid with a purity of 99.2% and was completely soluble in test medium at the concentrations tested. Preparation of the test solutions started with the highest concentration of 100 mg/L, applying a short period of magnetic stirring to accelerate dissolution of the test item in medium. The lower concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none, test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

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): 3 - 4 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 (Adjusted M2) medium (OECD 201 Guideline) was formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Acclimation period: Three to four days before the start of the test, fresh M2 medium was inoculated at a density of 1E4 cells/mL and was kept under the same conditions used in the test.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg/L as CaCO3
Test temperature:
between 21 and 23 °C
pH:
7.5 - 8.2
Nominal and measured concentrations:
Nominal: 0 mg/L (Control), 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L, and 100 mg/L
Measured: see Table 1
Results are based on nominal concentrations. Duplicate samples taken from all test concentrations at the start and the end of the test were analysed. The measured concentrations in samples both with and without algae were at the level of nominal throughout the test (i.e. 101-116% relative to nominal).

Details on test conditions:
TEST SYSTEM
- Test vessel: 40 mL glass, airtight, closed with no headspace to prevent loss of MTDID 49220 due to volatilization
- Fill volume: 40 mL
- Aeration: continuous shaking
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 221.5E+04 cells/mL
- No. of vessels per concentration: Three replicates of each test concentration, and one or two replicates of each test concentration without algae.
- No. of vessels per control: Six replicates
GROWTH MEDIUM
- Standard medium used:Standard OECD TG 201 test medium (M2) was adjusted for use in a sealed test vessel by increase of NaHCO3 to 300 mg/L and addition of 6 mM HEPES buffer, final pH 7.1± 0.3..
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 81 to 84 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 cuvettes (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
TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. three
RANGE-FINDING STUDY
- Combined limt/range-finding study
- Results used to determine the conditions for the definitive study:Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2CrO7)

Results and discussion

Effect concentrationsopen allclose all
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:
Effect parameters based on analytically confirmed nominal concentrations
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
72 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% confidence limit, 57 - 92 mg/L; effect parameters based on analytically confirmed nominal concentrations
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Effect parameter based on analytically confirmed nominal concentrations
Details on results:
- 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 algal cells exposed to 100 mg/L when compared to the control.
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
Results with reference substance valid? yes
- EC50: 1.6 mg/L (growth rate) with 95% confidence interval of 1.5 to 1.6 mg/L. 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 prior to the initiation of testing MTDID 49220.
Reported statistics and error estimates:
NOEC: Growth rate and yield data were normally distributed. NOEC determined using Williams Multiple Sequential t-test Procedure (α=0.05, one-sided, smaller).
EC10: Calculated using a 3-parameter normal cumulative distribution function (CDF) using non-linear regression analyses, with the percentages of growth rate inhibition versus the logarithms of the corresponding nominal concentrations of the test item (R-squared = 0.988, AIC = -140.207, Shapiro Wilk's normal distributiion of residuals p=0.128).
ErC50-value could be calculated for the growth rate (EC50 > maximum concentration tested).
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 2, Individual cell densities in the algal toxicity test (x10E+04 cells/mL)

Concentration (mg/L) Replicate  Time      
    0 h 24 h 48 h 72 h
0 (Control) 1 1.000 13.400 44.877 213.613
  2 1.000 8.312 42.550 229.280
  3 1.000 10.062 44.294 222.920
  4 1.000 9.479 43.288 213.102
  5 1.000 9.080 43.312 226.121
  6 1.000 9.104 44.871 224.032
Mean:   1.000 9.9 43.9 221.5
Std. Dev.:   0.0 1.8 1.0 6.7
CV:   0.0 18.2 2.2 3.0
1.0 1 1.000 8.253 40.902 199.226
  2 1.000 9.080 42.015 219.355
  3 1.000 9.919 42.961 228.786
Mean:   1.000 9.1 42.0 215.8
Std. Dev.:   0.0 0.8 1.0 15.1
CV:   0.0 9.2 2.5 7.0
3.2 1 1.000 8.301 42.122 220.307
  2 1.000 10.056 43.472 223.104
  3 1.000 8.402 46.644 236.296
Mean:   1.000 8.9 44.1 226.6
Std. Dev.:   0.0 1.0 2.3 8.5
CV:   0.0 11.0 5.3 3.8
10 1 1.000 9.276 39.551 211.150
  2 1.000 8.997 45.305 232.374
  3 1.000 8.878 41.527 226.805
Mean:   1.000 9.1 42.1 223.4
Std. Dev.:   0.0 0.2 2.9 11.0
CV:   0.0 2.3 6.9 4.9
32 1 1.000 10.080 38.147 211.311
  2 1.000 8.955 41.723 218.641
  3 1.000 8.759 44.454 224.609
Mean:   1.000 9.3 41.4 218.2
Std. Dev.:   0.0 0.7 3.2 6.7
CV:   0.0 7.7 7.6 3.1
100.0 1 1.000 12.454 22.361 52.683
  2 1.000 6.855 25.158 63.203
  3 1.000 8.592 25.967 64.524
Mean:   1.000 9.3 24.5 60.1
Std. Dev.:   0.0 2.9 1.9 6.5
CV:   0.0 30.8 7.7 10.8

Table 3, Growth rates (1/day) in the algal toxicity test

Concentration (mg/L) Replicate  Interval      
    0 - 24 h 24 - 48 h 48 - 72 h 0 - 72 h
0 (Control) 1 2.595 1.209 1.56 1.788
  2 2.118 1.633 1.684 1.812
  3 2.309 1.482 1.616 1.802
  4 2.249 1.519 1.594 1.787
  5 2.206 1.562 1.653 1.807
  6 2.209 1.595 1.608 1.804
Mean:   2.281 1.5 1.619 1.8
Std. Dev.:   0.1662 0.1524 0.0438 0.0101
CV:   7.285 10.161 2.707 0.561
1.0 1 2.111 1.601 1.583 1.765
  2 2.206 1.532 1.653 1.797
  3 2.294 1.466 1.672 1.811
Mean:   2.204 1.533 1.636 1.791
Std. Dev.:   0.092 0.0674 0.0469 0.0236
CV:   4.173 4.396 2.864 1.32
3.2 1 2.116 1.624 1.654 1.798
  2 2.308 1.464 1.636 1.803
  3 2.128 1.714 1.623 1.822
Mean:   2.184 1.601 1.638 1.808
Std. Dev.:   0.1074 0.1267 0.016 0.0124
CV:   4.917 7.915 0.98 0.689
10 1 2.227 1.45 1.675 1.784
  2 2.197 1.617 1.635 1.816
  3 2.184 1.543 1.698 1.808
Mean:   2.203 1.536 1.669 1.803
Std. Dev.:   0.0225 0.0834 0.0318 0.0166
CV:   1.021 5.425 1.905 0.921
32 1 2.311 1.331 1.712 1.784
  2 2.192 1.539 1.656 1.796
  3 2.17 1.624 1.62 1.805
Mean:   2.224 1.498 1.663 1.795
Std. Dev.:   0.0755 0.1509 0.0463 0.0102
CV:   3.396 10.076 2.786 0.568
100.0 1 2.522 0.585 0.857 1.321
  2 1.925 1.3 0.921 1.382
  3 2.151 1.106 0.91 1.389
Mean:   2.199 0.997 0.896 1.364
Std. Dev.:   0.3015 0.3697 0.0343 0.0372
CV:   13.707 37.073 3.832 2.726

The mean of the replicate coefficients of variation for section-by-section growth rate in the control was: 23%.

The coefficient of variation (CV) of the mean specific growth rate for the control from 0 to 72 h was 0.56%.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Control cell density increased by a factor of > 16 (i.e. 222); mean CV for section-by-section sp ecific growth rate in the controls was < 35% (i.e. 23%); CV of average specific growth rates in the whole test period for controls was < 7% (i.e. 0.56%).
Conclusions:
The toxicity of MTDID 49220 to Pseudokirchneriella subcapitata was evaluated according to OECD TG 201. The 72-hour ErC50 was found to be > 100 mg/L, the 72-hour ErC10 was 72 mg/L and the 72-hour NOEC was 32 mg/L.
Executive summary:

The toxicity of MTDID 49220 to Pseudokirchneriella subcapitata was examined in a static test conducted according to OECD TG 201. Test solutions were prepared as the control (0 mg/L), and at concentrations of 1.0, 3.2, 10, 32, and 100 mg/L (nominal). Nominal concentrations were verified with analysis. Test vessels were sealed, with no headspace due to the volatility of the MTDID 49220. The 72-hour ErC50 was found to be > 100 mg/L, the 72-hour ErC10 was 72 mg/L and the 72-hour NOEC was 32 mg/L. The test was conducted according to internationally accepted test guidelines and was GLP compliant. It is reliable without restriction and suitable for Risk Assessment, Classification and Labeling, and PBT Analysis.

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