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EC number: 701-003-6 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Toxicity to aquatic algae and cyanobacteria
Administrative data
Link to relevant study record(s)
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 28 November 2016 - 26 January 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Alga, Growth Inhibition Test)
- Version / remarks:
- Organization for Economic Co-operation and Development (OECD), OECD guidelines for Testing of Chemicals, guideline No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", Adopted March 23, 2006; Annex 5 corrected 28 July 2011.
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.3 (Algal Inhibition test)
- Version / remarks:
- Commission regulation (EC) No. 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C3: “Algal Inhibition Test”; Amended by EC No. 2016/266 of 7 December 2015, Publication No. L54.
- Deviations:
- no
- GLP compliance:
- yes
- Specific details on test material used for the study:
- Test item: 207258/AIdentification: MLA-3202Appearance: Clear amber-red liquidBatch: RC-1045Purity/Composition: UVCBTest item storage: At room temperature Stable under storage conditions until: 17 February 2019 (expiry date)Purity/composition correction factor: No correction factor requiredChemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)CAS Number: 1454803-04-3
- Analytical monitoring:
- yes
- Details on sampling:
- On the day of analysis, the test samples were thawed at room temperature. The samples were diluted in a 1:3 (v:v) ratio with methanol and analyzed. If necessary, the samples were further diluted with 75/25 (v/v) methanol/M2-medium to obtain concentrations within the calibration range.Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below. Frequency: at t=0 h, t=24 h and t=72 hVolume: 1.0 mLStorage : Samples were stored in a freezer (≤-15°C) until analysis.At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration 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 1.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis. Storage stability of samples under these conditions was demonstrated in project 511870.
- Vehicle:
- yes
- Details on test solutions:
- Preparation of test solutionsThe batch of MLA-3202 tested was a clear amber-red liquid and a UVCB substance. Test item was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. Preparation of test solutions started with the highest concentration of 100 mg/L in the combined limit/range-finding test and 15 mg/L in the full test. A one hour period of magnetic stirring was applied to accelerate the dissolving of the test item in the test medium. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colorless up to and including 15 mg/L. The concentration of 100 mg/L in the combined limit/range-finding test was slightly hazy, but considered completely dissolved as no Tyndall effect was observed when using laser pointer. 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 10^4 cells/mL.
- Test organisms (species):
- Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
- Details on test organisms:
- Species : Pseudokirchneriella subcapitata, strain: NIVA CHL 1Source : In-house laboratory culture.Reason for selection : This system is an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.
- Test type:
- static
- Water media type:
- freshwater
- Limit test:
- yes
- Total exposure duration:
- 72 h
- Hardness:
- Ca+Mg: 0.24 mmol/L (24 mg CaCO3/L)
- Test temperature:
- 21-24°C
- pH:
- 6.0-9.0
- Dissolved oxygen:
- Not specified
- Salinity:
- Not applicable
- Conductivity:
- Not applicable
- Nominal and measured concentrations:
- Combined limit/range-finding test Nominal concentrations: 0.10, 1.0 and 10 mg/L.Final test Nominal concentrations: 0.10, 0.35, 1.2, 4.3 and 15 mg/L
- Details on test conditions:
- Test duration: 72 hoursTest type: StaticTest vessels: 100 mL, all-glass, containing 50 mL of test solutionMedium: M2Cell density: An initial cell density of 1 x 104 cells/mL.Illumination: Continuously using TLD-lamps with a light intensity within the range of 95 to 96 µE.m-2.s-1.Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Reference substance (positive control):
- yes
- Remarks:
- potassium dichromate
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.19 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.19 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat.
- Basis for effect:
- cell number
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 0.16 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks on result:
- other: 95% confidence interval between: 0.13 - 0.20
- Duration:
- 72 h
- Dose descriptor:
- EC20
- Effect conc.:
- 0.66 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks on result:
- other: 95% confidence interval between: 0.56 - 0.76
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 9.8 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks on result:
- other: 95% confidence interval between: 8.6 - 11
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 0.17 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat.
- Basis for effect:
- cell number
- Remarks on result:
- other: 95% confidence interval between: 0.12 - 0.21
- Duration:
- 72 h
- Dose descriptor:
- EC20
- Effect conc.:
- 0.23 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat.
- Basis for effect:
- cell number
- Remarks on result:
- other: 95% confidence interval between: 0.18 - 0.27
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 0.43 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat.
- Basis for effect:
- cell number
- Remarks on result:
- other: 95% confidence interval between: 0.37 - 0.48
- Details on results:
- Combined limit/range-finding testMean cell densities, inhibition of growth rate and inhibition of yieldBased on these results samples taken from nominal concentrations of 0.10 and 100 mg/L were analyzed. The actual concentrations were at the level of nominal (94-102%) at the start of the exposure and at 5.7-98% of initial at the end of the test, respectively.Therefore, the expected EC50 for growth rate inhibition approximated the nominal concentration of 10 mg/L. The expected EC50 for yield inhibition was between 0.10 and 1.0 mg/L.All test conditions were maintained within the limits prescribed by the study plan.Final testMeasured test item concentrationsSamples taken from all concentrations were analyzed. The actual concentrations were at the level of nominal (88-97%). The measured concentrations were at the level of 31-90% of initially measured at the end of the exposure. The stability of the concentrations increased with the applied dose. The concentrations measured in the solution incubated with algae (nominally 1.2 mg/L) was more stable than its counterpart incubated with algae (78 vs. 45% of initial at the end of the test, respectively). This indicates that the observed decrease could be (at least partly) caused by adsorption to the growing algal biomass. Based on these results, the Time Weight Average (TWA) concentrations were calculated.Inhibition of growth rate and inhibition of yield.Growth rates were in the range of the controls at the two lowest concentrations tested during the 72-hour test period, whereas the growth rate of algae exposed to a TWA concentration of 0.68 mg/L and higher were increasingly reduced. Inhibition of yield increased with increasing concentration of MLA-3202 from 12% at TWA concentration of 0.19 mg/L upwards resulting in 93% inhibition at the highest concentration tested. Statistically significant inhibition of both growth rate and yield was found at the TWA concentrations of 0.68, 3.3 and 14 mg/L.Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.Experimental conditionsBelow shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit). During the exposure period the temperature measured in the incubator was maintained at 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).
- Results with reference substance (positive control):
- Potassium Dichromate significantly inhibited the growth rate of this fresh water algal species at nominal concentrations of 0.32 mg/L and higher.The EC50 for growth rate inhibition (72h-ERC50) was 1.3 mg/L with a 95% confidence interval ranging from 1.2 to 1.4 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.The EC50 for yield inhibition (72h-EYC50) was 0.45 mg/L with a 95% confidence interval ranging from 0.39 to 0.53 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.
- Validity criteria fulfilled:
- yes
- Conclusions:
- Under the conditions of the present study with Pseudokirchneriella subcapitata, MLA-3202 reduced growth rate and inhibited the yield of this fresh water algae species significantly at a TWA concentration of 0.68 mg/L and higher.The EC50 for growth rate inhibition (72h-ERC50) was 9.8 mg/L with a 95% confidence interval ranging from 8.6 to 11 mg/L.The EC50 for yield inhibition (72h-EYC50) was 0.43 mg/L with a 95% confidence interval ranging from 0.37 to 0.48 mg/L.The 72h-NOEC for both, growth rate and yield inhibition was 0.19 mg/L.
- Executive summary:
Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with MLA-3202.
The study procedures were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 2016/266, Part C.3, 2008; Amended by EC No. 2016/266.
The batch of MLA-3202 tested was a clear amber-red liquid and a UVCB substance. Test item was completely soluble in test medium at the concentrations tested.No correction was made for the purity/composition of the test item.
A final test was performed based on the results of a combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 0.10, 0.35, 1.2, 4.3 and 15 mg MLA-3202 per liter. The initial algal cell density was 104cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.
Samples taken from all concentrations were analyzed. The actual concentrations were at the level of nominal (88-97%). The measured concentrations were at the level of 31-90% of initially measured at the end of the exposure. The stability of the concentrations increased with the applied dose.
Based on these results, the Time Weight Average (TWA) concentrations were 0.035, 0.16, 0.68, 3.3 and 14 mg/L in nominally 0.10, 0.35, 1.2, 4.3 and 15 mg/L, respectively.
The study met the acceptability criteria prescribed by the study plan and was considered valid.
The effect parameters obtained in this study are summarized in the table below.
Parameter (mg/L)
NOEC
EC10
EC20
EC50
Growth rate
Value
0.19
0.16
0.66
9.8
lower 95%-cl
0.13
0.56
8.6
upper 95%-cl
0.20
0.76
11
Yield
Value
0.19
0.17
0.23
0.43
lower95%-cl
0.12
0.18
0.37
upper 95%-cl
0.21
0.27
0.48
Reference
Mean cell densities (x104cells/mL) during the combined limit/range-finding test
Time (h) | Nominal concentration of MLA-3202 (mg/L) | ||||
Control | 0.10 | 1.0 | 10 | 100 | |
0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
24 | 3.9 | n.d. | n.d. | n.d. | 1.5 |
48 | 21.6 | n.d. | n.d. | n.d. | 3.5 |
72 | 101.6 | 129.6 | 22.7 | 8.8 | 5.5 |
n.a. – not applicable
Percentage inhibition of growth rate during the combined limit/range-finding test
MLA-3202 Nominal conc. (mg/L) | Mean | Std. Dev. | n | %Inhibition |
Control | 1.537 | 0.0560 | 6 | |
0.10 | 1.617 | 0.0687 | 3 | -5.2 |
1.0 | 1.040 | 0.0155 | 3 | 32.3 |
10 | 0.725 | 0.0369 | 3 | 52.8 |
100 | 0.558 | 0.0792 | 6 | 63.7 |
Percentage inhibition of yield during the combined limit/range-finding test
MLA-3202 Nominal conc. (mg/L) | Mean | Std. Dev. | n | %Decrease |
Control | 100.6 | 15.90 | 6 | |
0.10 | 128.6 | 27.73 | 3 | -27.8 |
1.0 | 21.7 | 1.04 | 3 | 78.5 |
10 | 7.8 | 0.95 | 3 | 92.2 |
100 | 4.5 | 1.18 | 6 | 95.6 |
Measured concentrations versus nominal concentrations
MLA-3202 Nominal concentration (mg/L) | Measured concentration (mg/L) | TWA concentration (mg/L) | ||
t=0h | t=24h | t=72 h | ||
0.10 | 0.0877 | 0.0279 | 0.0269 | 0.035 |
0.35 | 0.339 | 0.207 | 0.103 | 0.19 |
1.2 | 1.12 | 0.680 | 0.507 | 0.68 |
1.2 WA | 1.16 | 0.946 | 0.903 | 0.97 |
4.3 | 4.07 | 3.35 | 2.76 | 3.3 |
15 | 14.6 | 14.1 | 13.2 | 14 |
Percentage inhibition of growth rate (total test period) during the final test
MLA-3202 TWA conc. (mg/L) | Mean | Std. Dev. | n | %Inhibition |
Control | 1.693 | 0.0563 | 6 | |
0.035 | 1.721 | 0.0561 | 3 | -1.6 |
0.19 | 1.652 | 0.0188 | 3 | 2.5 |
0.68 | 1.242* | 0.0389 | 3 | 26.7 |
3.3 | 0.977* | 0.0370 | 3 | 42.3 |
14 | 0.859* | 0.0160 | 3 | 49.3 |
Percentage inhibition of growth rate at different time intervals during the final test
MLA-3202 TWA conc. (mg/L) | n | 0 – 24 h | 24 – 48 h | 48 – 72h | |||
Mean | %Inhibition | Mean | %Inhibition | Mean | %Inhibition | ||
Control | 6 | 1.514 | 0.0 | 1.843 | 0.0 | 1.723 | 0.0 |
0.035 | 3 | 1.274 | 15.9 | 2.151 | -16.8 | 1.737 | -0.9 |
0.19 | 3 | 0.880 | 41.9 | 2.326 | -26.2 | 1.749 | -1.6 |
0.68 | 3 | 0.839 | 44.6 | 1.931 | -4.8 | 0.956 | 44.5 |
3.3 | 3 | 0.772 | 49.0 | 1.778 | 3.5 | 0.382 | 77.8 |
14 | 3 | 1.134 | 25.1 | 0.849 | 53.9 | 0.595 | 65.5 |
Percentage inhibition of yield during the final test
MLA-3202 TWA conc. (mg/L) | Mean | Std. Dev. | n | %Decrease |
Control | 161.6 | 28.44 | 6 | |
0.035 | 175.3 | 28.50 | 3 | -8.4 |
0.19 | 141.0 | 7.95 | 3 | 12.8 |
0.68 | 40.7* | 4.69 | 3 | 74.8 |
3.3 | 17.8* | 2.02 | 3 | 89.0 |
14 | 12.2* | 0.63 | 3 | 92.5 |
* - effect was statistically significant
pH levels recorded during the final test
MLA-3202 TWA conc. (mg/L) | pH | |
t=0h | t=72h | |
Control | 8.1 | 7.9 |
0.035 | 8.0 | 7.8 |
0.19 | 8.0 | 7.7 |
0.68 | 8.0 | 7.7 |
3.3 | 8.0 | 7.8 |
14 | 8.0 | 7.8 |
Individual cell densities (x104cells/mL)
Time | Replicate | TWA concentration of MLA-3202 (mg/L) | |||||
Control | 0.035 | 0.19 | 0.68 | 3.3 | 14 | ||
0 h | 1 2 3 4 5 6 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
2 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | |
3 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | |
4 | 1.000 |
|
|
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| |
5 | 1.000 |
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| |
6 | 1.000 |
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| |
n: |
| 6 | 3 | 3 | 3 | 3 | 3 |
Mean: |
| 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
Std.Dev.: |
| 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
CV: |
| 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
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24 h | 1 2 3 4 5 6 | 2.750 | 3.250 | 2.000 | 2.000 | 2.250 | 5.000 |
2 | 6.000 | 3.750 | 2.000 | 2.250 | 1.500 | 3.000 | |
3 | 5.000 | 3.750 | 3.500 | 2.750 | 3.000 | 2.000 | |
4 | 4.750 |
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5 | 6.000 |
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6 | 3.750 |
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n: |
| 6 | 3 | 3 | 3 | 3 | 3 |
Mean: |
| 4.7 | 3.6 | 2.5 | 2.3 | 2.3 | 3.3 |
Std.Dev.: |
| 1.3 | 0.3 | 0.9 | 0.4 | 0.8 | 1.5 |
CV: |
| 27.2 | 8.1 | 34.6 | 16.4 | 33.3 | 45.8 |
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48 h | 1 2 3 4 5 6 | 17.330 | 28.000 | 22.250 | 13.250 | 13.000 | 7.500 |
2 | 25.000 | 30.500 | 27.250 | 12.500 | 15.000 | 6.000 | |
3 | 26.500 | 34.000 | 24.750 | 24.500 | 10.750 | 8.500 | |
4 | 35.750 |
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5 | 31.250 |
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6 | 43.500 |
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n: |
| 6 | 3 | 3 | 3 | 3 | 3 |
Mean: |
| 29.9 | 30.8 | 24.8 | 16.8 | 12.9 | 7.3 |
Std.Dev.: |
| 9.1 | 3.0 | 2.5 | 6.7 | 2.1 | 1.3 |
CV: |
| 30.5 | 9.8 | 10.1 | 40.1 | 16.5 | 17.2 |
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72 h | 1 2 3 4 5 6 | 136.500 | 183.750 | 143.250 | 44.500 | 20.000 | 13.250 |
2 | 153.000 | 144.750 | 149.250 | 36.250 | 20.000 | 13.750 | |
3 | 151.500 | 200.250 | 133.500 | 44.250 | 16.500 | 12.500 | |
4 | 192.000 |
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5 | 138.750 |
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6 | 204.000 |
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n: |
| 6 | 3 | 3 | 3 | 3 | 3 |
Mean: |
| 162.6 | 176.3 | 142.0 | 41.7 | 18.8 | 13.2 |
Std.Dev.: |
| 28.4 | 28.5 | 7.9 | 4.7 | 2.0 | 0.6 |
CV: |
| 17.5 | 16.2 | 5.6 | 11.3 | 10.7 | 4.8 |
According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16. In the current test it was found to be 163. The test fulfils this validity criterion.
Individual growth rates (day-1)
Time | Replicate | TWA concentration of MLA-3202 (mg/L) | |||||
Control | 0.035 | 0.19 | 0.68 | 3.3 | 14 | ||
0-24 h | 1 2 3 4 5 6 | 1.012 | 1.179 | 0.693 | 0.693 | 0.811 | 1.609 |
| 2 | 1.792 | 1.322 | 0.693 | 0.811 | 0.405 | 1.099 |
| 3 | 1.609 | 1.322 | 1.253 | 1.012 | 1.099 | 0.693 |
| 4 | 1.558 |
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| 5 | 1.792 |
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| 6 | 1.322 |
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Mean: |
| 1.514 | 1.274 | 0.88 | 0.839 | 0.772 | 1.134 |
Std.Dev.: |
| 0.3016 | 0.0826 | 0.3231 | 0.161 | 0.3482 | 0.4592 |
n: |
| 6 | 3 | 3 | 3 | 3 | 3 |
CV: |
| 19.9 | 6.5 | 36.7 | 19.2 | 45.1 | 40.5 |
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0-48 h | 1 2 3 4 5 6 | 1.426 | 1.666 | 1.551 | 1.292 | 1.282 | 1.007 |
| 2 | 1.609 | 1.709 | 1.653 | 1.263 | 1.354 | 0.896 |
| 3 | 1.639 | 1.763 | 1.604 | 1.599 | 1.187 | 1.07 |
| 4 | 1.788 |
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| 5 | 1.721 |
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| 6 | 1.886 |
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Mean: |
| 1.678 | 1.713 | 1.603 | 1.385 | 1.275 | 0.991 |
Std.Dev.: |
| 0.1594 | 0.0487 | 0.0507 | 0.1864 | 0.0836 | 0.0882 |
n: |
| 6 | 3 | 3 | 3 | 3 | 3 |
CV: |
| 9.5 | 2.8 | 3.2 | 13.5 | 6.6 | 8.9 |
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0-72 h | 1 2 3 4 5 6 | 1.639 | 1.738 | 1.655 | 1.265 | 0.999 | 0.861 |
| 2 | 1.677 | 1.658 | 1.669 | 1.197 | 0.999 | 0.874 |
| 3 | 1.674 | 1.767 | 1.631 | 1.263 | 0.934 | 0.842 |
| 4 | 1.752 |
|
|
|
|
|
| 5 | 1.644 |
|
|
|
|
|
| 6 | 1.773 |
|
|
|
|
|
|
|
|
|
|
|
|
|
Mean: |
| 1.693 | 1.721 | 1.652 | 1.242 | 0.977 | 0.859 |
Std.Dev.: |
| 0.0563 | 0.0561 | 0.0188 | 0.0389 | 0.037 | 0.016 |
n: |
| 6 | 3 | 3 | 3 | 3 | 3 |
CV: |
| 3.3 | 3.3 | 1.1 | 3.1 | 3.8 | 1.9 |
The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 3.3%. According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7%.The test fulfils this validity criterion.
Section-by-section growth rate(day-1)
Time | Replicate | TWA concentration of MLA-3202 (mg/L) | |||||
Control | 0.035 | 0.19 | 0.68 | 3.3 | 14 | ||
0 - 24 h h | 1 2 3 4 5 6 | 1.012 | 1.179 | 0.693 | 0.693 | 0.811 | 1.609 |
| 2 | 1.792 | 1.322 | 0.693 | 0.811 | 0.405 | 1.099 |
| 3 | 1.609 | 1.322 | 1.253 | 1.012 | 1.099 | 0.693 |
| 4 | 1.558 |
|
|
|
|
|
| 5 | 1.792 |
|
|
|
|
|
| 6 | 1.322 |
|
|
|
|
|
|
|
|
|
|
|
|
|
Mean: |
| 1.514 | 1.274 | 0.88 | 0.839 | 0.772 | 1.134 |
Std.Dev.: |
| 0.3016 | 0.0826 | 0.3231 | 0.161 | 0.3482 | 0.4592 |
n: |
| 6 | 3 | 3 | 3 | 3 | 3 |
CV: |
| 19.9 | 6.5 | 36.7 | 19.2 | 45.1 | 40.5 |
|
|
|
|
|
|
| |
24 - 48 h | 1 2 3 4 5 6 | 1.841 | 2.154 | 2.409 | 1.891 | 1.754 | 0.405 |
| 2 | 1.427 | 2.096 | 2.612 | 1.715 | 2.303 | 0.693 |
| 3 | 1.668 | 2.205 | 1.956 | 2.187 | 1.276 | 1.447 |
| 4 | 2.018 |
|
|
|
|
|
| 5 | 1.65 |
|
|
|
|
|
| 6 | 2.451 |
|
|
|
|
|
|
|
|
|
|
|
|
|
Mean: |
| 1.843 | 2.151 | 2.326 | 1.931 | 1.778 | 0.849 |
Std.Dev.: |
| 0.3581 | 0.0543 | 0.3358 | 0.2387 | 0.5136 | 0.5378 |
n: |
| 6 | 3 | 3 | 3 | 3 | 3 |
CV: |
| 19.4 | 2.5 | 14.4 | 12.4 | 28.9 | 63.4 |
|
|
|
|
|
|
| |
48 - 72 h | 1 2 3 4 5 6 | 2.064 | 1.881 | 1.862 | 1.211 | 0.431 | 0.569 |
| 2 | 1.812 | 1.557 | 1.701 | 1.065 | 0.288 | 0.829 |
| 3 | 1.743 | 1.773 | 1.685 | 0.591 | 0.428 | 0.386 |
| 4 | 1.681 |
|
|
|
|
|
| 5 | 1.491 |
|
|
|
|
|
| 6 | 1.545 |
|
|
|
|
|
|
|
|
|
|
|
| |
Mean: |
| 1.723 | 1.737 | 1.749 | 0.956 | 0.382 | 0.595 |
Std.Dev.: |
| 0.2058 | 0.165 | 0.0981 | 0.3242 | 0.082 | 0.2229 |
n: |
| 6 | 3 | 3 | 3 | 3 | 3 |
CV: |
| 11.9 | 9.5 | 5.6 | 33.9 | 21.4 | 37.5 |
The mean of the replicate coefficients of variation for section-by-section growth rate in the control was: 18%. According to OECD 201, the mean coefficient of variation, measured in the control from 0 to 72 h, must not be higher than 35%.The test fulfils this validity criterion.
Individual yields(x104cells/mL)
Time | Replicate | TWA concentration of MLA-3202 (mg/L) | |||||
Control | 0.035 | 0.19 | 0.68 | 3.3 | 14 | ||
24 h | 1 2 3 4 5 6 | 1.8 | 2.3 | 1 | 1 | 1.3 | 4 |
| 2 | 5 | 2.8 | 1 | 1.3 | 0.5 | 2 |
| 3 | 4 | 2.8 | 2.5 | 1.8 | 2 | 1 |
| 4 | 3.8 |
|
|
|
|
|
| 5 | 5 |
|
|
|
|
|
| 6 | 2.8 |
|
|
|
|
|
|
|
|
|
|
|
|
|
Mean: |
| 3.7 | 2.6 | 1.5 | 1.3 | 1.3 | 2.3 |
Std.Dev.: |
| 1.28 | 0.29 | 0.87 | 0.38 | 0.75 | 1.53 |
n: |
| 6 | 3 | 3 | 3 | 3 | 3 |
CV: |
| 34.5 | 11.2 | 57.7 | 28.6 | 60 | 65.5 |
|
|
|
|
|
|
| |
48 h | 1 2 3 4 5 6 | 16.3 | 27 | 21.3 | 12.3 | 12 | 6.5 |
| 2 | 24 | 29.5 | 26.3 | 11.5 | 14 | 5 |
| 3 | 25.5 | 33 | 23.8 | 23.5 | 9.8 | 7.5 |
| 4 | 34.8 |
|
|
|
|
|
| 5 | 30.3 |
|
|
|
|
|
| 6 | 42.5 |
|
|
|
|
|
|
|
|
|
|
|
|
|
Mean: |
| 28.9 | 29.8 | 23.8 | 15.8 | 11.9 | 6.3 |
Std.Dev.: |
| 9.11 | 3.01 | 2.5 | 6.72 | 2.13 | 1.26 |
n: |
| 6 | 3 | 3 | 3 | 3 | 3 |
CV: |
| 31.5 | 10.1 | 10.5 | 42.7 | 17.8 | 19.9 |
|
|
|
|
|
|
| |
72 h | 1 2 3 4 5 6 | 135.5 | 182.8 | 142.3 | 43.5 | 19 | 12.3 |
| 2 | 152 | 143.8 | 148.3 | 35.3 | 19 | 12.8 |
| 3 | 150.5 | 199.3 | 132.5 | 43.3 | 15.5 | 11.5 |
| 4 | 191 |
|
|
|
|
|
| 5 | 137.8 |
|
|
|
|
|
| 6 | 203 |
|
|
|
|
|
|
|
|
|
|
|
|
|
Mean: |
| 161.6 | 175.3 | 141 | 40.7 | 17.8 | 12.2 |
Std.Dev.: |
| 28.44 | 28.5 | 7.95 | 4.69 | 2.02 | 0.63 |
n: |
| 6 | 3 | 3 | 3 | 3 | 3 |
CV: |
| 17.6 | 16.3 | 5.6 | 11.5 | 11.3 | 5.2 |
STATISTICS: GROWTH RATE
Test for normality of distribution
Shapiro-Wilk´s Test on Normal Distribution
Treatm. [mg/L] | Mean | s | n |
Control | 1.693 | 0.0563 | 6 |
0.035 | 1.721 | 0.0561 | 3 |
0.19 | 1.652 | 0.0188 | 3 |
0.68 | 1.242 | 0.0389 | 3 |
3.3 | 0.977 | 0.0370 | 3 |
14 | 0.859 | 0.0160 | 3 |
Results:
Number of residuals = 19
Shapiro-Wilk´s W = 0.964
p(W) = 0.645
p(W) is greater than the selected significance level of 0.010; thus treatment data do not
significantly deviate from normal distribution.
Test for homogeneity of variance
Levene´s Test on Variance Homogeneity (with Residuals)
Source | SS | df | MSS | F | p(F) |
Treatment | 0.00385 | 5 | 0.00077 | 2.330 | 0.094 |
Residuals | 0.00496 | 15 | 0.00033 |
|
|
Total | 0.00880 | 20 |
|
|
|
The Levene test indicates variance homogeneity (p > 0.010).
Variance homogeneity check passed (p > 0.01).
Normal-distribution and variance-homogeneity requirements are fulfilled.
A parametric multiple test is advisable.
Determination of NOEC
The analysis of contrasts revealed a linear trend, thus the selected Williams test was performed.
Williams Multiple Sequential t-test Procedure
Treatm. [mg/L] | Mean | s | df | LhM | %MDD | t | t* | Sign. |
Control | 1.693 | 0.0441 |
| |||||
0.035 | 1.721 | 0.0441 | 15 | 1.721 | -3.2 | 0.89 | -1.75 | - |
0.19 | 1.652 | 0.0441 | 15 | 1.652 | -3.4 | -1.33 | -1.83 | - |
0.68 | 1.242 | 0.0441 | 15 | 1.242 | -3.4 | -14.48 | -1.85 | + |
3.3 | 0.977 | 0.0441 | 15 | 0.977 | -3.4 | -22.97 | -1.86 | + |
14 | 0.859 | 0.0441 | 15 | 0.859 | -3.4 | -26.77 | -1.87 | + |
+: significant; -: non-significant
A NOEC of 0.19 mg/L is suggested by the program.
STATISTICS: YIELD
Shapiro-Wilk´s Test on Normal Distribution
Treatm. [mg/L] | Mean | s | n |
Control | 161.6 | 28.44 | 6 |
0.035 | 175.3 | 28.50 | 3 |
0.19 | 141.0 | 7.95 | 3 |
0.68 | 40.7 | 4.69 | 3 |
3.3 | 17.8 | 2.02 | 3 |
14 | 12.2 | 0.63 | 3 |
Results:
Number of residuals = 20
Shapiro-Wilk´s W = 0.936
p(W) = 0.204
p(W) is greater than the selected significance level of 0.010; thus treatment data do not
significantly deviate from normal distribution.
Test for homogeneity of variance
Levene´s Test on Variance Homogeneity (with Residuals)
Source | SS | df | MSS | F | p(F) |
Treatment | 2098.453 | 5 | 419.691 | 6.029 | 0.003 |
Residuals | 1044.247 | 15 | 69.616 |
|
|
Total | 3142.700 | 20 |
|
|
|
The Levene test indicates variance heterogeneity (p <= 0.010)
Variance homogeneity check failed
However, normal distribution requirements are fulfilled.
The Welch-t-test for non-homogeneous variances with Bonferroni-Holm-adjustment is advisable.
Determination of NOEC
Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm
Treatm. [mg/L] | Mean | s | df | %MDD | t | p(t) | Alpha(i) | Sign. |
Control | 161.6 | 28.44 |
| |||||
0.035 | 175.3 | 28.50 | 4 | -26.6 | 0.68 | 0.732 | 0.050 | - |
0.19 | 141.0 | 7.95 | 6 | -18.9 | -1.65 | 0.075 | 0.025 | - |
0.68 | 40.7 | 4.69 | 5 | -21.5 | -10.15 | < 0.001 | 0.017 | + |
3.3 | 17.8 | 2.02 | 5 | -22.8 | -12.32 | < 0.001 | 0.013 | + |
14 | 12.2 | 0.63 | 5 | -24.2 | -12.87 | < 0.001 | 0.010 | + |
+: significant; -: non-significant
A NOEC of 0.19 mg/L is suggested by the program.
Calibration Curves
Calibration curves were constructed using five concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration2weighting factor. The coefficient of correlation (r) was > 0.99 for each curve.
QC Samples
During the range-finding test, the chromatograms of the blank QC samples showed no peak at the retention time of the test item. During the final test, a small response at the retention time of the test item was detected in the chromatograms of the blank QC samples. Concentration was below the limit of detection which was 0.00008 mg/L during the range-finding test and 0.00007 mg/L during the final test.
During the range-finding test, the mean accuracy of the 100 mg/L QC samples was slightly above the criterion range of 70 - 110% (i.e. 113% of target). The mean accuracy of the 0.01 mg/L QC samples was within the criterion range. During the final test, the mean accuracies of QC samples containing test item fell within the criterion of 70-110%. It demonstrated that the analytical method was adequate for the determination of the test item concentration in the test samples.
Test Samples
During the final test, a small response at the retention time was observed in the chromatograms of nominal 0 mg/L samples. Concentration was just above the limit of detection.
QC Samples
Date of preparation | Date of | Concentration | Accuracy | |||
Target | Nominal | Analyzed | Individual | Mean | ||
07 Dec 2016 | 07 Dec 2016 | 0 | 0.00 | < LOD | n.a. | n.a. |
|
|
| 0.00 | < LOD | n.a. |
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|
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|
|
07 Dec 2016 | 07 Dec 2016 | 0.01 | 0.0100 | 0.0108 | 108 | 107 |
|
|
| 0.0100 | 0.0107 | 107 | (n.a.) |
|
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|
|
|
|
07 Dec 2016 | 07 Dec 2016 | 100 | 100 | 112 | 112 | 113 |
|
|
| 100 | 107 | 107 | (4) |
|
|
| 100 | 119 | 119 |
|
|
|
| 100 | 114 | 114 |
|
|
|
| 100 | 116 | 116 |
|
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|
|
|
|
|
|
26 Jan 2017 | 26 Jan 2017 | 0 | 0.00 | < LOD | n.a. | n.a. |
|
|
| 0.00 | < LOD | n.a. |
|
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|
|
|
|
|
|
26 Jan 2017 | 26 Jan 2017 | 0.01 | 0.0100 | 0.0103 | 103 | 103 |
|
|
| 0.0100 | 0.0103 | 103 |
|
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|
|
|
|
|
|
26 Jan 2017 | 26 Jan 2017 | 100 | 100 | 97.5 | 98 | 100 |
|
|
| 100 | 102 | 102 |
|
LOD The limit of detection of the method, taking a dilution factor of four into account, was determined to be 0.00008 mg/L on 07 Dec 2016 and 0.00007 mg/L on 26 Jan 2017.
n.d. Not detected.
n.a. Not applicable.
Range-finding Test: Test Samples
Time of sampling | Date of sampling | Date of | Concentration | Relative to nominal | Relative to initial | |
Nominal | Analyzed | |||||
0 | 28 Nov 2016 | 07 Dec 2016 | 0.1 | 0.0940 | 94 |
|
|
|
| 100 | 102 | 102 |
|
|
|
|
|
|
|
|
24 | 29 Nov 2016 | 07 Dec 2016 | 0.1 | 0.0176 | 18 | 19 |
|
|
| 100 | 108 | 108 | 106 |
|
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|
|
|
|
|
72 | 01 Dec 2016 | 07 Dec 2016 | 0.1 | 0.00532 | 5.3 | 5.7 |
|
|
| 100 | 100 | 100 | 98 |
1 Samples were stored in the freezer (≤ -15°C) until the day of analysis.
2 Estimated value, calculated by extrapolation of the calibration curve.
Final Test: Test Samples
Time of sampling | Date of sampling | Date of | Concentration | Relative to nominal | Relative to initial | |
Nominal | Analyzed | |||||
0 | 16 Jan 2017 | 26 Jan 2017 | 0 | 0.0000893 |
|
|
|
|
| 0.1 | 0.0877 | 88 |
|
|
|
| 0.35 | 0.339 | 97 |
|
|
|
| 1.2 | 1.12 | 94 |
|
|
|
| 1.22 | 1.16 | 97 |
|
|
|
| 4.3 | 4.07 | 95 |
|
|
|
| 15 | 14.6 | 97 |
|
|
|
|
|
|
|
|
24 | 17 Jan 2017 | 26 Jan 2017 | 0 | 0.0000843 |
|
|
|
|
| 0.1 | 0.0279 | 28 | 32 |
|
|
| 0.35 | 0.207 | 59 | 61 |
|
|
| 1.2 | 0.680 | 57 | 60 |
|
|
| 1.22 | 0.946 | 79 | 82 |
|
|
| 4.3 | 3.35 | 78 | 82 |
|
|
| 15 | 14.1 | 94 | 96 |
|
|
|
|
|
|
|
72 | 19 Jan 2017 | 26 Jan 2017 | 0 | 0.0000953 |
|
|
|
|
| 0.1 | 0.0269 | 27 | 31 |
|
|
| 0.35 | 0.103 | 30 | 31 |
|
|
| 1.2 | 0.507 | 42 | 45 |
|
|
| 1.22 | 0.903 | 75 | 78 |
|
|
| 4.3 | 2.76 | 64 | 68 |
|
|
| 15 | 13.2 | 88 | 90 |
1 Samples were stored in the freezer (≤ -15°C) until the day of analysis.
2 Without algae.
3 Estimated value, calculated by extrapolation of the calibration curve.
n.a. Not applicable.
Description of key information
Key value determined in GLP accredited laboratory study according to the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 2016/266, Part C.3, 2008; Amended by EC No. 2016/266.
Key value for chemical safety assessment
- EC50 for freshwater algae:
- 0.43 mg/L
- EC10 or NOEC for freshwater algae:
- 0.19 mg/L
Additional information
The batch of MLA-3202 tested was a clear amber-red liquid and a UVCB substance. Test item was completely
soluble in test medium at the concentrations tested.No correction was made for the purity/composition of the test item.
A final test was performed based on the results of a combined limit/range-finding test. Six exponentially growing
algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 0.10, 0.35, 1.2, 4.3 and 15 mg MLA-3202 per liter. The initial algal cell density was 104cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.
Samples taken from all concentrations were analyzed. The actual concentrations were at the level of nominal (88-97%). The measured concentrations were at the level of 31-90% of initially measured at the end of the exposure. The stability of the concentrations increased with the applied dose.
Based on these results, the Time Weight Average (TWA) concentrations were 0.035, 0.16, 0.68, 3.3 and 14 mg/L in nominally 0.10, 0.35, 1.2, 4.3 and 15 mg/L, respectively.
The study met the acceptability criteria prescribed by the study plan and was considered valid.
The effect parameters obtained in this study are summarized in the table below.
Parameter (mg/L) |
NOEC |
EC10 |
EC20 |
EC50 |
|
Growth rate |
Value |
0.19 |
0.16 |
0.66 |
9.8 |
lower 95%-cl |
|
0.13 |
0.56 |
8.6 |
|
upper 95%-cl |
|
0.20 |
0.76 |
11 |
|
Yield |
Value |
0.19 |
0.17 |
0.23 |
0.43 |
lower95%-cl |
|
0.12 |
0.18 |
0.37 |
|
upper 95%-cl |
|
0.21 |
0.27 |
0.48 |
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.