Decan-5-olide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 2.5, 5.0, 10, 20, 40, and 80 mg/L
- Sample storage conditions before analysis: test samples were analysed immediately after sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test chemical was directly dissolved in the OECD medium (500mg of test chemical was dissolved in 500 ml of OECD medium to get final concenttration of 1g/L)
- Control: OECD medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Source (laboratory, culture collection): Sterile, unicellular, suspension cultures of algae were obtained from Denmark Technical University, Denmark
- Age of inoculum (at test initiation): The algal inocula for a test was taken from an exponentially-growing pre-culture and mixed with the OECD medium
- Method of cultivation: Algae was maintained in the OECD medium in the orbital shaker, at temperature 22 ± 2°C, as per the recomeded light intesity as per OECD 201.

ACCLIMATION
- Culturing media and conditions (same as test or not): Similar to test
- Any deformed or abnormal cells observed: No abnormal cells were found

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21°C to 22.2°C

pH:

6.9 to 7.4

Nominal and measured concentrations:

Nominal concentrations: 2.5, 5.0, 10, 20, 40, and 80 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Type (delete if not applicable): loosely closed
- Material, size, headspace, fill volume: 100 ml conical flasks filled with 60 ml was used for the study, with 40 ml head space
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 10000 cells/ml
- Control end cells density: 26833 cells/ml
- No. of organisms per vessel: 60 ml X 10000 cells/ml
- No. of vessels per concentration (replicates): 2 replicates
- No. of vessels per control (replicates): 3 replicates
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes ( OECD medium)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(3000-4000 Lux)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: The cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae with the help of heamocytometer
- Chlorophyll measurement: Not applicable
- Other: Not applicable

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: Not applicable, test species was used as per the recommendations of OECD 201 guidelines
- Range finding study: Main study concentrations were selected based on the available data
- Test concentrations: Not applicable
- Results used to determine the conditions for the definitive study: Not applicable

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 (Potassium Dichromate)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

26.542 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI - 21.90 and 32.66 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No morphological abnormalities
- Unusual cell shape: None
- Colour differences: NOne
- Adherence to test vessels: None
- Aggregation of algal cells: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes, Valid
- EC50: 1.732 mg/L

Reported statistics and error estimates:

quantitative concentration-response relationship by regression analysis, was done using SysSTAT Pro software to obtain EC50 values.

Validity criteria fulfilled:

yes

Conclusions:

The median effective concentration of test chemical for the fresh water algae ( P. subcapitata) was reported to be 26.54 mg/L with 95% CI is 21.90 to 32.66 mg/L.

Executive summary:

Toxicity to aquatic algae study was carried out. The study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test). Pseudokirchneriella subcapitata (green algae) obtained from the Denmark Technical University, Denmark was used as a test algae. The algal inocula for a test was taken from an exponentially-growing pre-culture and mixed with the OECD medium. Algae was maintained in the OECD medium in the orbital shaker, at temperature 22 ± 2°C, as per the recomeded light intesity as per OECD 201. Test chemical was directly dissolved in the OECD medium (500mg of test chemical was dissolved in 500 ml of OECD medium to get final concenttration of 1g/L). Test chemical conc. used for the study were 2.5, 5.0, 10, 20, 40, and 80 mg/l, respectively. Test samples were analysed immediately after sampling by using GC-MS. Test algae were exposed to different test chemical conc. in a conical flasks. Conical flasks of 100 ml was filled with 60 ml which was loosely closed and it has with 40 ml head space. Aeration was not proviided not during the study. Intial cell density of the test algae was 10000 cells/ml. OECD medium was used as a standard test medium during the study. Test conditions involve a temperature of 21°C to 22.2°C, pH of 6.9 to 7.4 with a continuous, uniform fluorescent illumination(3000-4000 Lux) under a photoperiod of 16 Hour Light Period : 8 Hour Dark Period.  The cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae with the help of heamocytometer. All test experiments were performed in duplicates and control experiments were performed in triplicates, respectively. K2Cr2O7 (Potassium Dichromate) was used as a reference substance. The EC50 value of the reference substance Potassium Dichromate was determined to be 1.732 mg/L. Quantitative concentration-response relationship by regression analysis, was done using SysSTAT Pro software to obtain EC50 values. Test validity criterion has been fulfilled during the study. On the basis of the effect on growth rate of the test algae, the 96 hr EC50 value was determined to be 26.54 mg/L (95% CI  is 21.90 to 32.66 mg/L). Thus, test chemical was considered as toxic to aquatic algae at environmental relevant concentrations and hence, considered to be classified in ''aquatic chronic category 3'' as per the CLP classification criteria.

Description of key information

Toxicity to aquatic algae:

Toxicity to aquatic algae study was carried out. The study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test). Pseudokirchneriella subcapitata (green algae) obtained from the Denmark Technical University, Denmark was used as a test algae. The algal inocula for a test was taken from an exponentially-growing pre-culture and mixed with the OECD medium. Algae was maintained in the OECD medium in the orbital shaker, at temperature 22 ± 2°C, as per the recomeded light intesity as per OECD 201. Test chemical was directly dissolved in the OECD medium (500mg of test chemical was dissolved in 500 ml of OECD medium to get final concenttration of 1g/L). Test chemical conc. used for the study were 2.5, 5.0, 10, 20, 40, and 80 mg/l, respectively. Test samples were analysed immediately after sampling by using GC-MS. Test algae were exposed to different test chemical conc. in a conical flasks. Conical flasks of 100 ml was filled with 60 ml which was loosely closed and it has with 40 ml head space. Aeration was not proviided not during the study. Intial cell density of the test algae was 10000 cells/ml. OECD medium was used as a standard test medium during the study. Test conditions involve a temperature of 21°C to 22.2°C, pH of 6.9 to 7.4 with a continuous, uniform fluorescent illumination(3000-4000 Lux) under a photoperiod of 16 Hour Light Period : 8 Hour Dark Period.  The cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae with the help of heamocytometer. All test experiments were performed in duplicates and control experiments were performed in triplicates, respectively. K2Cr2O7 (Potassium Dichromate) was used as a reference substance. The EC50 value of the reference substance Potassium Dichromate was determined to be 1.732 mg/L. Quantitative concentration-response relationship by regression analysis, was done using SysSTAT Pro software to obtain EC50 values. Test validity criterion has been fulfilled during the study. On the basis of the effect on growth rate of the test algae, the 96 hr EC50 value was determined to be 26.54 mg/L (95% CI  is 21.90 to 32.66 mg/L). Thus, test chemical was considered as toxic to aquatic algae at environmental relevant concentrations and hence, considered to be classified in ''aquatic chronic category 3'' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

26.54 mg/L

Additional information

Experimental studies of the test chemical and supporting study for its functionally similar read across chemical were reviewed for toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

 

Toxicity to aquatic algae study was carried out. The study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test). Pseudokirchneriella subcapitata (green algae) obtained from the Denmark Technical University, Denmark was used as a test algae. The algal inocula for a test was taken from an exponentially-growing pre-culture and mixed with the OECD medium. Algae was maintained in the OECD medium in the orbital shaker, at temperature 22 ± 2°C, as per the recomeded light intesity as per OECD 201. Test chemical was directly dissolved in the OECD medium (500mg of test chemical was dissolved in 500 ml of OECD medium to get final concenttration of 1g/L). Test chemical conc. used for the study were 2.5, 5.0, 10, 20, 40, and 80 mg/l, respectively. Test samples were analysed immediately after sampling by using GC-MS. Test algae were exposed to different test chemical conc. in a conical flasks. Conical flasks of 100 ml was filled with 60 ml which was loosely closed and it has with 40 ml head space. Aeration was not proviided not during the study. Intial cell density of the test algae was 10000 cells/ml. OECD medium was used as a standard test medium during the study. Test conditions involve a temperature of 21°C to 22.2°C, pH of 6.9 to 7.4 with a continuous, uniform fluorescent illumination(3000-4000 Lux) under a photoperiod of 16 Hour Light Period : 8 Hour Dark Period.  The cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae with the help of heamocytometer. All test experiments were performed in duplicates and control experiments were performed in triplicates, respectively. K2Cr2O7 (Potassium Dichromate) was used as a reference substance. The EC50 value of the reference substance Potassium Dichromate was determined to be 1.732 mg/L. Quantitative concentration-response relationship by regression analysis, was done using SysSTAT Pro software to obtain EC50 values. Test validity criterion has been fulfilled during the study. On the basis of the effect on growth rate of the test algae, the 96 hr EC50 value was determined to be 26.54 mg/L (95% CI  is 21.90 to 32.66 mg/L). Thus, test chemical was considered as toxic to aquatic algae at environmental relevant concentrations and hence, considered to be classified in ''aquatic chronic category 3'' as per the CLP classification criteria.

 

Another toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (Experimental study report. 2018). The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution 100 mg/l was prepared by dissolving test chemical in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 2.5, 5.0, 10, 20, 40 and 80 mg/l, respectively. Study was performed in a static system for 72 hrs. Desmodesmus subspicatus were exposed to test chemical in 50 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessel containing OECD medium without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. The 24 hr EC50 value of the reference substance was determined to be 0.77 mg/l. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 4.5% and the specific growth rate in the control was 1.77 per day, indicating that the biomass in the control cultures have increased exponentially by a factor of more than 16 within the 72 hr exposure duration. Thus, the validity criterion of the test has been fulfilled. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 20.5 mg/l (95% CL: 10.7 to 39.3 mg/l). Thus, based on the EC50 value, test chemical can be considered to be toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

 

For the test chemical, an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance with the Scenedesmus cell proliferation inhibition test, DIN 38412 part 9.Test chemical concentrations were not verified analytically. Test chemical conc. used for the study were 0 (control), 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 mg/l (nominal conc.). Study was performed using Scenedesmus subspicatus as a test organism in a static system in quadruplicates (for test conc.). Initial cell concentration used for the study was 10000 cells/ml. Test organisms were exposed to test chemical in 250 mL Erlenmeyer flask. The test vessels were placed at a temperature of 19.85°C under continuous illumination with a light intensity of 6.2 miS/cm. Cell growth was measured fluorometrically in all flasks at 24, 48, 72 and 96 hours of incubation period. On the basis of effect on growth rate of the test organism Scenedesmus subspicatus, the 96 hr EC20, ErC50 and EC90 was determined to be 20, 79 and > 500 mg/l (nominal conc.), respectively. Thus, based on this, chemical was considered as toxic to aquatic algae and hence, considered to be classified in ‘aquatic chronic category 3’ as per the CLP classification criteria. 

 

On the basis of the above results, it can be concluded that the test chemical can be considered as toxic to aquatic algae and considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.