Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

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:
Experimental start date: 13 September 2017 Experimental completion date:
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene bis[1,3-dihydro-1,3-dioxoisobenzofuran-5-carboxylate]
EC Number:
217-062-1
EC Name:
Ethylene bis[1,3-dihydro-1,3-dioxoisobenzofuran-5-carboxylate]
Cas Number:
1732-96-3
Molecular formula:
C20H10O10
IUPAC Name:
2-(1,3-dioxo-1,3-dihydro-2-benzofuran-5-carbonyloxy)ethyl 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxylate
impurity 1
Chemical structure
Reference substance name:
Benzene-1,2,4-tricarboxylic acid 1,2-anhydride
EC Number:
209-008-0
EC Name:
Benzene-1,2,4-tricarboxylic acid 1,2-anhydride
Cas Number:
552-30-7
Molecular formula:
C9H4O5
IUPAC Name:
Benzene-1,2,4-tricarboxylic acid 1,2-anhydride
impurity 2
Chemical structure
Reference substance name:
not assignable
IUPAC Name:
not assignable
Specific details on test material used for the study:
Identification: RIKACID TMEG-500
Physical state/Appearance: Cream colored powder
Batch: 5259
Purity: 81.1%
Expiry Date: 24 April 2018
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Range-Finding Test
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. The sample bottles were fortified with 200 µL of formic acid prior to the addition of the test sample. All 0-Hour samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Preliminary Stability Analyses
The analysis of the range-finding test preparations failed. Therefore, in order to determine the stability of the test item under test conditions a 100% v/v saturated solution of the test item was prepared and a dilution of the 100% v/v saturated solution was made after filtration to give a further stock solution of 10% v/v saturated solution. A sample of the 10 and 100% v/v saturated solution test preparations was taken for immediate analysis in a bottle fortified with 200 µL of formic acid. A further sample of each test concentration was then incubated under test conditions for a period of 72 hours prior to analysis.

Definitive Test
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All 0-Hour samples were stored frozen prior to analysis whilst the 72-Hour test preparations were analyzed on the day. The 0-Hour sample bottles were fortified with 200 µL of formic acid prior to the addition of the samples. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

Range-Finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 14 mg/L could be obtained using a saturated solution method of preparation.
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (6.6 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorious Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 56, 32, 18 and 10% v/v saturated solution. An aliquot (500 mL) of each of the stock solutions was separately inoculated with 6.3 mL of algal suspension to give the required test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 ºC.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 ºC until the algal cell density was approximately 10^4 - 10^5 cells/mL.

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Study design

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

Test conditions

Test temperature:
24 ± 1 ºC
pH:
6.3 - 8.2
A concentration dependent decrease in pH was observed in the test cultures at 0 hours in the range of pH 7.8 at 1.8 mg/L through to pH 6.5 at 18 mg/L. This was considered to be due to an intrinsic property of the test item and as such no adjustment was made to the pH of the test preparations prior to inoculation with algal cells.
Nominal and measured concentrations:
Range-finding test
Nominal test concentrations of 0.10, 1.0, 10 and 100% v/v.

Definitive Test
Based on the results of the range-finding test the following nominal test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100% v/v saturated solution.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.8 to 18 mg/L. There was no significant change in the measured concentrations at 72 hours (1.9 to 18 mg/L) and so the results are based on 0-Hour measured test concentrations only.
Details on test conditions:
Range-Finding Test
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Definitive Test
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control and three flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 4.00 x 10^5 cells per mL. Inoculation of 500 mL of test medium with 6.3 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate. The positive control was conducted between 06 November 2017 and 09 November 2017.

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
13 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
Range-finding Test
The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution. However, growth was observed to be reduced at 100% v/v saturated solution.
Based on this information test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution were selected for the definitive test.
Whilst samples were taken from the range-finding test in order to determine the stability of the test item under test conditions the analysis failed.

Preliminary Stability Analyses
Analysis of the 10 and 100% v/v saturated solution test preparations at 0 hours (see Annex 5) showed measured test concentrations of 1.8 and 17 mg/L respectively were obtained. There was no significant decrease in the measured test concentrations at 72 hours indicating that the test item was stable under the conditions of the test.

Definitive Test

Growth Data
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

Accordingly the following results were determined from the data:
Inhibition of Growth Rate
ErC10 (0 - 72 h): 14 mg/L
ErC20 (0 - 72 h): 15 mg/L
ErC50 (0 - 72 h): 16 mg/L; 95% confidence limits 16 - 17 mg/L

Where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control, 1.8, 3.2, 5.7 and 10 mg/L test concentrations (P≥0.05), however the 18 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 10 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 18 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h): 11 mg/L
EyC20 (0 - 72 h): 12 mg/L
EyC50 (0 - 72 h): 13 mg/L; 95% confidence limits 13 - 14 mg/L

Where EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences between the control, 1.8, 3.2, 5.7 and 10 mg/L test concentrations (P≥0.05), however the 18 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 10 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 18 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 367 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours: 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours: 1.84 x 10^6 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 13% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Water Quality Criteria
Temperature was maintained at 24 ± 1 ºC throughout the test.
The pH value of the control cultures was determined to be pH 7.8 at 0 and 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
A concentration dependent decrease in pH was observed in the test cultures at 0 hours in the range of pH 7.8 at 1.8 mg/L through to pH 6.5 at 18 mg/L. This was considered to be due to an intrinsic property of the test item and as such no adjustment was made to the pH of the test preparations prior to inoculation with algal cells.

Observations on Test Item Solubility
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 1.8, 3.2, 5.7 and 10 mg/L test cultures were observed to be green dispersions whilst the 18 mg/L test cultures were observed to be clear colorless solutions.

Results with reference substance (positive control):
A positive control (Envigo Study Number RD71YQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h): 1.6 mg/L; 95% confidence limits 1.4 – 1.8 mg/L
EyC50 (0 – 72 h): 0.77 mg/L; 95% confidence limits 0.68 – 0.87 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results based on the 0-Hour measured test concentrations:

EC50 (mg/L) 95% Confidence Limits (mg/L) NOEC (mg/L) LOEC (mg/L)
Growth Rate 16 16 - 17 10 18
Yield 13 13 - 14 10 18

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

 Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 14 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding test, Pseudokirchneriella subcapitatawas exposed to solutions of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Results

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.8 to 18 mg/L. There was no significant change in the measured concentrations at 72 hours (1.9 to 18 mg/L) and so the results are based on 0-Hour measured test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable

EC50(mg/L)

95% Confidence Limits (mg/L)

No Observed Effect Concentration (NOEC) (mg/L)

Lowest Observed Effect Concentration (LOEC) (mg/L)

Growth Rate

16

16

-

17

10

18

Yield

13

13

-

14

10

18