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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from secondary source
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
yes
Remarks:
dimethyl formamide was used as a vehicle.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22°C
pH:
8.0 – 8.1 (at start), 7.1 – 9.5 (at end)
Nominal and measured concentrations:
Test chemical conc. used for the study were 0 (control); 0 (solvent control (dimethyl formamide)); 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 mg/l, respectively.
Details on test conditions:
TEST SYSTEM
- Initial cells density: 10000 cells/ml
- No. of vessels per concentration (replicates): Triplicates
- No. of vessels per control (replicates): 6 replicates
- No. of vessels per vehicle control (replicates): Triplicates

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: 7000 lux


Reference substance (positive control):
not specified
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
7.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
< 2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
14.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
At all test concentrations, growth of the algae was delayed.
Validity criteria fulfilled:
not specified
Conclusions:
Based on the effect of test chemical on growth rate of test algae, the 48 & 72 hr EC50 value was determined to be 7.3 and 14.3 mg/l, respectively. On the basis of the effect on biomass, the 48 & 72 hr EC50 value was determined to be <2.5 and 2.9 mg/l, respectively.
Executive summary:

Toxicity to aquatic algae study was carried out for 72 hrs. Study was performed following the principles of the OECD Guideline 201 (Alga, Growth Inhibition Test). Pseudokirchneriella subcapitata (green algae) was used as a test algae. Test chemical analysis was carried out using gas chromatography with flame ionization detection. Initial recovery (0 hours) ranged from 38 – 91 % and ranged from 0.5 – 7.0 % after 24 hours Dimethyl formamide was used as a vehicle. Test chemical conc. used for the study were 0 (control); 0 (solvent control (dimethyl formamide)); 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 mg/l, respectively. Initial cell density of test algae was 10000 cells/ml. Study was performed under static conditions. Test conditions involve a pH of 8.0 – 8.1 (at start), 7.1 – 9.5 (at end); temperature of 22°C under a continuous photoperiod and a light intensity of 7000 lux. All test treatment and vehicle control system were performed in triplicates whereas control system were performed in 6 replcates. At all test concentrations, growth of the algae was delayed. Based on the effect of test chemical on growth rate of test algae, the 48 & 72 hr EC50 value was determined to be 7.3 and 14.3 mg/l, respectively. On the basis of the effect on biomass, the 48 & 72 hr EC50 value was determined to be <2.5 and 2.9 mg/l, respectively. Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic algae. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per the CLP classification criteria.

Description of key information

Toxicity to aquatic algae study was carried out for 72 hrs (Secondary source, 2008). Study was performed following the principles of the OECD Guideline 201 (Alga, Growth Inhibition Test). Pseudokirchneriella subcapitata (green algae) was used as a test algae. Test chemical analysis was carried out using gas chromatography with flame ionization detection. Initial recovery (0 hours) ranged from 38 – 91 % and ranged from 0.5 – 7.0 % after 24 hours Dimethyl formamide was used as a vehicle. Test chemical conc. used for the study were 0 (control); 0 (solvent control (dimethyl formamide)); 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 mg/l, respectively. Initial cell density of test algae was 10000 cells/ml. Study was performed under static conditions. Test conditions involve a pH of 8.0 – 8.1 (at start), 7.1 – 9.5 (at end); temperature of 22°C under a continuous photoperiod and a light intensity of 7000 lux. All test treatment and vehicle control system were performed in triplicates whereas control system were performed in 6 replcates. At all test concentrations, growth of the algae was delayed. Based on the effect of test chemical on growth rate of test algae, the 48 & 72 hr EC50 value was determined to be 7.3 and 14.3 mg/l, respectively. On the basis of the effect on biomass, the 48 & 72 hr EC50 value was determined to be <2.5 and 2.9 mg/l, respectively. Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic algae. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
14.3 mg/L

Additional information

Various experimental studies and predicted data of the test chemical and its read across chemical were reviewed for toxicity to aquatic algae end point which are summarized as below:

 

In an experimental study from secondary source (2008), toxicity to aquatic algae study was carried out for 72 hrs. Study was performed following the principles of the OECD Guideline 201 (Alga, Growth Inhibition Test). Pseudokirchneriella subcapitata (green algae) was used as a test algae. Test chemical analysis was carried out using gas chromatography with flame ionization detection. Initial recovery (0 hours) ranged from 38 – 91 % and ranged from 0.5 – 7.0 % after 24 hours Dimethyl formamide was used as a vehicle. Test chemical conc. used for the study were 0 (control); 0 (solvent control (dimethyl formamide)); 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 mg/l, respectively. Initial cell density of test algae was 10000 cells/ml. Study was performed under static conditions. Test conditions involve a pH of 8.0 – 8.1 (at start), 7.1 – 9.5 (at end); temperature of 22°C under a continuous photoperiod and a light intensity of 7000 lux. All test treatment and vehicle control system were performed in triplicates whereas control system were performed in 6 replcates. At all test concentrations, growth of the algae was delayed. Based on the effect of test chemical on growth rate of test algae, the 48 & 72 hr EC50 value was determined to be 7.3 and 14.3 mg/l, respectively. On the basis of the effect on biomass, the 48 & 72 hr EC50 value was determined to be <2.5 and 2.9 mg/l, respectively. Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic algae. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per the CLP classification criteria.

 

Another toxicity to aquatic algae study was carried out for 72 hrs. Study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test). Test was performed under static conditions. On the basis of the effect of test chemical on growth rate of test algae, the 72 hr NOEC and EC50 value was determined to be 0.33 and 1.9 mg/l, respectively. Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic algae. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per the CLP classification criteria.

 

Another acute toxicity to aquatic algae was carried out for assessing the effect of test chemical. Study was performed following the principles of the OECD TG 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test).  Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen / Germany) was used as a test algae. The algae were cultivated in RCC’s laboratories under standardized conditions according to the test guidelines. Due to the limited solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was prepared by mixing 109.2 mg of the test item into 1090 mL of test water using intense stirring. The stirring vessel was completely filled and sealed by a glass stopper. The dispersion was stirred for 24 hours at room temperature in the dark in order to dissolve a maximum amount of the test item in the dispersion. After the stirring period, the dispersion was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm) and the undiluted filtrate (saturated solution) was used as highest test concentration and as stock solution for the preparation of the lower concentrated test media. The negative pressure of the filtration unit was reduced as much as possible to avoid losses of the test item during filtration. For the preparation of the test media of the lower test concentrations, the filtrate was diluted with test water. The test media were prepared just before the start of the test. Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing with modifications according to the International Standard ISO 14442 [2]. The modifications were made to improve the growth conditions for the algae in a closed test system.The concentration of NaHCO3 in the test water was increased by 200 mg/L to 250 mg/L (as carbon source for the algal growth), and 6 mmol/L HEPES-buffer were added to keep the pH in the test media during the test period as constant as possible.Analytical measurements of test chemical conc. were done by using HPLC analysis with UV/Vis detection. For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control. For sampling at the end of the test, the test medium of the treatment replicates was pooled. Additionally, one flask of test medium of the dilution 1:10 was incubated without algae under test conditions in order to document possible adsorption of the test item onto the algae. Immediately after sampling, the samples were diluted 1:1 (v/v) with acetonitrile in order to stabilize the samples during the storage period. All samples were stored at about -20 °C until analysis. In pre-experiments (non-GLP), the test item proved to be stable in the test water under these storage conditions.The concentrations of the test chemical were determined in the duplicate test medium samples from the dilutions of 1:32, 1:10, 1:3.2 and the undiluted filtrate. The samples from the dilution of 1:100 were not analyzed, since this concentration was below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times. A saturated solution (filtrate) and the dilutions 1:3.2, 1:10, 1:32 and 1:100 of the saturated solution were tested. Additionally, a control was tested in parallel (test water without test item). The enlarged spacing factor of 3.2 between the test concentrations was chosen, as the concentration-effect relationship was flat according to the results of the range-finding test. Thus, a wide concentration range had to be tested. 50-mL Erlenmeyer flasks were used per replicate. Each test flask was filled with approximately 60 mL algal suspension and sealed with a glass stopper to prevent loss of the volatile test item. The test flasks were labeled with the RCC study number and all necessary additional information to ensure unique identification. During the test, the test solutions were continuously stirred by magnetic stirrers. The test design included three replicates per test concentration and six replicates of the control. The test was started using a nominal algal cell density of 10000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter®, Model ZM). The initial cell density corresponded to 1.1 x 10E3 relative fluorescence units. The conversion factor between algal cell density and fluorescence signal was therefore 9.0 cells/mL per fluorescence unit. A static test design in a closed test system was applied. The duration of the test was 72 hours. Potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test condition. The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK® Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate. At the end of the test, a sample was taken from the control and from a test concentration with reduced algal growth (dilution 1:3.2). The shape and size of the algal cells were visually inspected. This test concentration was chosen, since the algal cell density was too low for a reliable examination at the highest test concentration. During the test period of 72 hours, the highest inhibitory effect on the growth of the algae was determined during the first day of the test. A lag phase was determined in the algal cultures exposed to the test item. During the second and third day of the test, a recovery of the algal growth was determined in the test item treatments with the exception of the highest test concentration in which the algal growth was completely inhibited. The lag phase in the exposed algal cultures may indicate recovery after initial toxic stress or reduced exposure due to loss of the test item (although the test was performed in a closed test system). Therefore, the algal growth inhibition determined during the first day of the test was taken into account for the evaluation of the study and the biological results were related to the initial measured concentrations of the test item in the test media.The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing in the dilution of 1:3.2 and the algal cells in the control. The test item did not affect the shape and size of the algal cells up to at least this concentration. In the control the biomass increased by a factor of 242 over 72 hours (>16). The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates, during 72 hours was 17%. (< 35%). The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.3%. (< 7%). The EC10, EC20 and EC50 values for the inhibition of growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis. For the determination of the LOEC and NOEC, growth rate and yield at the test concentrations were compared to the control values by Dunnett’s tests. Only effect concentration at 24h were derived due to technical impossibilities and lack of detectable levels of test substance at 72h. The 72 hr EC50 value of the reference substance was determined to be 1.2 mg/l. The 24 hr EC50 value of the test chemical was determined to be 1.9 mg/l. Thus, test chemical was considered as toxic to aquatic algae.

 

In a supporting weight of evidence study frompeer reviewed journal (A.M. Apia et. al., 2019), toxicity to aquatic algae study was carried out for 72 hrs. Study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test). Test was performed under static conditions. On the basis of the effect of test chemical on growth rate of test algae, the 24 hr EC10 value was determined to be 0.79 mg/l. Thus, based on the EC50 value, test chemical was considered as toxic to aquatic algae.

 

For the test chemical, toxicity to aquatic algae study was carried out for 72 hrs (PPDB database, 2021). Pseudokirchneriella subcapitata (green algae) was used as a test organism. Test was performed under static conditions. On the basis of the effect of test chemical on growth rate of test algae, the 72 hr EC50 value was determined to be >0.25 mg/l. Thus, based on the EC50 value, test chemical was considered as toxic to aquatic algae.

 

In a prediction done using the EPI Suite ECOSAR version 1.11, the toxicity of the test chemical to aquatic algae was predicted. On the basis of effect of test chemical observed in a static system on the growth rate of the test organism during the 96 hr exposure duration, the median effect concentration (EC50) for the test chemical was estimated to be 3.978 mg/l. Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic algae. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per the CLP classification criteria.

 

Additional toxicity to aquatic algae study was carried out for 72 hrs. Study was performed following the OECD Guideline 201 (Alga, Growth Inhibition Test). Test was performed under static conditions. Based on the effect of test chemical on growth rate of test algae, the 72 hr NOEC and EC50 value was determined to be 0.56 and 0.028 mg/l, respectively. On the basis of the effect on area under the growth curve (AUG), the 72 hr NOEC and EC50 value was determined to be 0.72 and 0.09 mg/l, respectively. Thus, based on the EC50 value, test chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic acute category 1/chronic category 1' as per the CLP classification criteria.

 

On the basis of the above results, it can be concluded that the test chemical was considered as toxic to aquatic algae.