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Ecotoxicological information

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:
key study
Study period:
31 Oct 2021 to 02 Dec 2021
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:
OECD Guideline 201. "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", Adopted March 23, 2006; Annex 5 corrected 28 July 2011.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
No further details specified in the atudy report
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from the limit concentration and the control according to the schedule below. A method was developed for four major components of the UVCB test material.
Frequency: at t=0 h and t=72 h.
Volume: 0.80 mL from the approximate centre of the test solutions.
Storage: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
At the end of the exposure period, the replicates with algae were pooled for the limit concentration, and for the control before sampling.
Vehicle:
no
Details on test solutions:
The batch of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid tested was a clear light yellow liquid UVCB. The test material was visually not completely soluble in test medium at the loading rate prepared.
Due to the expected very low solubility of the test material in test medium, a WAF was prepared at a loading rate of 1.0 mg/L, which was expected to be far above the solubility limit of the test material. A loading rate of 1.0 mg/L represents the lowest amount of test material that can be weighed and allows for visual confirmation of the presence of undissolved test material.
The test material was weighed out on a watch glass, after which the watch glass was added to the test medium. A one-day period of slow magnetic stirring was applied. This slow stirring ensured maximum dissolution of the test material in medium, while attempting to avoid the formation of micelles. The one-day period of stirring was chosen since no test material could be measured in the non-GLP solubility trial, and therefore no Saturation Test could be conducted to confirm the most suitable test solution preparation. The stirring time was chosen to be long enough for maximum dissolution, but short enough to prevent potential degradation of the test material over a longer period.
During stirring the vortex depth was minimal, i.e. formed a dimple. This slow stirring ensured maximum dissolution of the test material in medium, while attempting to avoid the formation of micelles. The obtained mixture was allowed to settle for a period of 3 hours. After this period of settlement, test material was still observed on the watch glass, and in addition, some droplets were floating on the surface of the test solution. This indicates that even at the low loading rate of 1.0 mg/L the test material was not fully dissolved.
Thereafter, the WSF was collected by means of siphoning from the approximate centre of the solution and was inspected on appearance and presence of undissolved material by using a laser-pen (i.e. presence of the Tyndall effect). All test solutions were clear and colourless at the end of the preparation procedure. No undissolved material was observed in the test solutions.
Pre-incubation of test vessels was applied. To this end, volumes of 50 mL test solution were added to each replicate of the test concentration. The vessels were then pre-incubated for one day to minimize the risk of test material loss due to potential sorption of test material to the glassware. At the end of the pre-incubation phase, the test vessels were emptied and re-filled with freshly prepared volumes of 50 mL of the respective test solution. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10E4 cells/mL.
Any residual volumes were discarded.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Raphidocelis subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is a 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
Remarks on exposure duration:
In accordance with the test guideline
Post exposure observation period:
No post exposure observation period specified in the study report
Hardness:
Hardness (Ca+Mg): 0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
between 21 and 23°C
pH:
8.0 - 8.1
Dissolved oxygen:
Not specified
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
WSF prepared at loading rates of 1.0 mg/L.
Details on test conditions:
Testing Strategy and Experimental Design
It was chosen to perform a limit test, as no toxicity was expected due to the low solubility of the test material in water.
Two limit tests were performed in the present study.
The first limit test did not meet the validity criteria (mean coefficient of variation for section-by-section specific growth rates was > 35%) and thus had to be repeated. Both the analytical and biological results were comparable to those of the second limit test. The results of the final limit test are reported below.

Test Concentrations
Test material: WSF prepared at loading rates of 1.0 mg/L.
Control: Test medium without test material or other additives.
Replicates:
6 replicates of the control
6 replicates of the limit concentration
2 replicates of the limit concentration without algae.

Test Procedure and Conditions
Test duration: 72 hours
Test type: Static
Test vessels: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
Test Medium: M2
Cell density: An initial cell density of 1 x 1e4 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 84 to 88 µE.m-2.s-1.
Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Measurements and Recordings
pH: At the beginning and at the end of the test, for the limit concentration and the control
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: At the end of the final test microscopic observations were performed on the limit concentration and the control to observe for any abnormal appearance of the algae.

Recording of Cell Densities
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 immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicate, without algae, as background for the treated solutions.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WSF
Basis for effect:
yield
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WSF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WSF
Basis for effect:
yield
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WSF
Basis for effect:
growth rate
Details on results:
Measured Test Material Concentrations
An analytical method was developed for four major compounds of the test material, and samples were analysed based on each of these compounds.
Samples taken from the limit concentration and the control were analysed. At the start of the test, a test material related response was observed, indicating exposure. However, the measured concentrations of the whole test material based on each of the four components were below the Limit of Quantification (LOQ) of 2.0 µg/L. At the end of the test the concentrations were also below the LOQ, though this response was probably not test item related.
Based on these results, effect parameters were expressed based on loading rates.

Inhibition of Growth Rate and Inhibition of Yield
No significant differences were recorded between the values for growth rate or yield at the limit concentration when compared to the control group.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the limit concentration when compared to the control.

Experimental Conditions
The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 unit).
During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C. Temperature remained within the limits prescribed by the study plan (21 24°C, constant within ±1°C).
Results with reference substance (positive control):
Reference Test
Test Facility Study No. 20326772
Experimental Start Date: 27 Aug 2021
Experimental Completion Date: 02 Sep 2021
The batch of Raphidocelis subcapitata tested, showed expected sensitivity to Potassium dichromate based on the historical range of reference tests performed by the Test Facility in the last ten years.
The raw data from this study are kept in the Charles River Den Bosch archives. The test described above was performed non-GLP.
Reported statistics and error estimates:
For determination of the NOELR and the ELx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the limit concentration compared with those obtained in the control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test, α=0.05, one-sided, smaller). This statistical comparison was preceded by a check on normal distribution of the data (Shapiro-Wilk’s Test) and a test for homogeneity of the variances (Levene’s test).
The ELx-values could not be determined using regression analysis, since a limit test was performed.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Growth Rate and Percentage Inhibition for the Total Test Period




















Test material1


Loading rate (mg/L)



Mean



Std. Dev.



n



%Inhibition



Control


1.0



1.776


1.762



0.0185


0.0139



6


6



 


0.77



 


Growth rate and Percentage Inhibition at Different Time Intervals































Test meterial1


Loading rate (mg/L)



n



0 – 24 h



24 – 48 h



48 – 72 h



Mean



%Inhibition



Mean



%Inhibition#



Mean



%Inhibition#



Control


1.0



6


6



1.987


1.917



 


3.5



1.771


1.789



 


-1.0



1.569


1.580



 


-0.70



# Negative values indicate stimulation rather than inhibition


 


Yield and Percentage Inhibition for the Total Test Period




















Test material1


Loading rate (mg/L)



Mean



Std. Dev.



n



%Inhibition



Control


1.0



205.104


196.678



11.5645


8.1891



6


6



 


4.10



 


Effect Parameters




















Parameter (mg/L)



NOELR



EL10



EL20



EL50



Growth rate


Yield



1.0


1.0



>1.0


>1.0



>1.0


>1.0



>1.0


>1.0



 


pH Levels Recorded during the Test



















Test material1


Loading rate (mg/L)



pH



T=0h



T=72h



Control


1.0



8.0


8.0



8.1


8.1



 


1 Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

Validity criteria fulfilled:
yes
Remarks:
The study met the validity criteria prescribed bu the study plan and was considered valid.
Conclusions:
The 72h-EL10, EL20 and EL50 values for inhibition of growth rate (ERCX) and yield (EYCX) exceeded the maximum solubility of the test material in test medium, i.e. exceeded a loading rate of 1.0 mg/L.
Executive summary:

The objective of the study was to evaluate Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid for its ability to generate toic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOELR, EL10, EL20 and EL50 for both inhibition of growth rate and inhibition of yield.


A limit test was performed based on he results of several pre-tests.


Test conditions and results of the limit test are presented below:


 


Test material: Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid


Appearance: Clear light yellow liquid


Purity: UVCB


Preparation of test solutions: WSF prepared at a loading rate of 1.0 mg/L


Specific procedure: Test vessels were pre-incubated for one day to minimise the risk of test material loss due to potential sorption of test material to the glass ware.


 


Experimental set-up


Guideline: OECD TG 201 and OECD GD 23


Test concentration: WSF prepared at a loading rate of 1.0 mg/L


Control: Blank control


Number of replicates: 6 replicates per control group and for the limit concentration.


Sampling for analysis: at t=0 and t=72 h.


 


Experimental conditions


pH and temperature: Within the ranges specified in OECD TG 201


light regime: Continuous


 


Results


Actual exposure concentrations: The concentration of the test material based on the four major compounds for which a method was developed were measure, however, at all time points these were below the LOQ or 2 μg/L.


Recorded effects: No statistically significant inhibition of algal growth rate and yield was recorded at the limit concentration.


 


The effect parameters (based on loading rates) obtained in the study are summarised in the table below.




















Parameter (mg/L)



NOELR



EL10



EL20



EL50



Growth rate


Yield



1.0


1.0



>1.0


>1.0



>1.0


>1.0



>1.0


>1.0



 


In conclusion, under the conditions of the present study with Raphidocelis subcapitata, no inhibition of growth rate or inhibition of yield was recorded at the limit concentration of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid tested.


The 72h-EL10, EL20 and EL50 values for inhibition of growth rate (ERCX) and yield (EYCX) exceeded the maximum solubility of the test material in test medium, i.e. exceeded a loading rate of 1.0 mg/L.


Due to the very low solubility of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid in test medium, concentration levels that might be toxic for algae could not be reached.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
April 29, 2003 to Ausgut 18, 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP. The study is read across to an analogous substance; refer to image and further information below.
Justification for type of information:
In accordance with Regulation (EC) 1907/2006 Annex XI (1.5) and the relevant ECHA guidance documents, the substance is read across to reduce the need for unnecessary repeat testing on the basis that the substances are similar on the basis of:
1) a common functional group and
2) the common precursors and/or the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals.

The molecules share a similar route of manufacture and similar starting materials, some of which are common to each molecule.
Additional details and justification is attached in Section 13. A further confirmatory study is currently underway on the substance.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
GLP compliance:
yes
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.22 - 0.79 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: cell growth inhibition and growth rate reduction

Description of key information

 Read across to structural analogue. All members of this category of esters have a hydrophobic nature. Based on structural similarities of the substance of interest and the members of the category it was concluded that it was justified to apply read across for this endpoint. The 72h-EC50 exceeded the maximum solubility of the analogue in the test medium.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information

Read across to structural analogue. All members of this category of esters have a hydrophobic nature. Based on structural similarities of the substance of interest and the members of the category it was concluded that it was justified to applyread across for this endpoint.

 

Selenastrum capricornutum, Fresh Water Algal Growth Inhibition Test with HATCOL 3331.

A limit test was performed exposing exponentially growing algal cultures to a filtered (ca. 5 μm) HATCOL 3331 solution prepared at a loading rate of 100 mg/I and a blank-control. The initial cell density was 104cells/mL. The total test period was 72 hours. Samples for determination of actual exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test period.

 

Analysis of the samples showed that average exposure concentrations were at or above the solubility limit of HATCOL 3331 (i.e. <0.2 mg/I).

HATCOL 3331 induced no inhibition of cell growth or reduction of growth rate in Selenastrum capricornutum at the concentration obtained in a filtered solution prepared at a loading rate of 100 mg/l, corresponding to an average exposure concentration of 0.22-0.79 mg/I (NOEC).

Due to the very low solubility of HATCOL 3331 in water, concentration levels that might be toxic for algae could not be reached.