4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

WoE report is based on two short term toxicity study of aquatic algae for the test chemical :
This study was designed to access the toxic effects of the test chemical on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

2.Short term toxicity of test material was evaluated on aquatic algae

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

not specified

Details on test solutions:

The test solution was prepared in aseptic condition. The test substance was prepared by adding 25.767 µl of test substance in 250 ml of BBM to get the final concentration of 103.07 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment.

Test organisms (species):

other: 2. Chlorella vulgaris 3. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Source (laboratory, culture collection): National Environmental Engineering Research Institute (NEERI), Nagpur (Laboratory)
- Method of cultivation: Bold’s Basal Medium(BBM)
ACCLIMATION
- Culturing media and conditions (same as test or not): The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

24 ° C ± 2°C
2.23±2°C

pH:

6.5 - 7
sample at concentration 20.0 mg.l'1: pH = 7.9 changed to pH = 7.8 during the test
control : pH = 8.2 changed to pH = 7.7 during the test
control 1 + acetone: pH = 7.9 changed to pH = 7.7 during the test
control 2 + acetone: pH = 7.9 changed to pH = 7.7 during the test

Nominal and measured concentrations:

2 mg/l, 4.4 mg/l, 9.68 mg/l, 21.296 mg/l, 46.8512 mg/l and 103.07 mg/l nominal concentrations were used in the study. All the six concentration were in geometric series spaced by a factor of 2.
2.0.5 , 1.3 , 3.2 , 8.0 , 20.0 mg/l

Details on test conditions:

1.
TEST SYSTEM
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: 100 ml conical flasks filled with 60 ml was used for the study.
- Initial cells density: 10000cells/ml
- No. of organisms per vessel: 10000cells/ml
- No. of vessels per concentration (replicates): Two replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control
GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(1500Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer - The absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.
- Chlorophyll measurement: No data
- Other: 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 (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: All the six concentrations were in geometric series spaced by a factor of 2.
- Test concentrations: Six test concentration were: 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l,100mg/l and 200mg/l (Nominal concentrations)
- Results used to determine the conditions for the definitive study: Mortality of test organisms
Other:
Incubation :
1. The temperature of the orbital shaking incubator was kept constant throughout the period of exposure of the experiment. The temperature was maintained at 24 ° C ±2°C.
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (1500Lux).
3. The pH of the control cultures needs to be noted during the study and the pH of the control medium should not increase by more than 1.5 units during the test.
4. The orbital shaking incubator was set at a speed of 120 revolutions per minute throughout the study period. This is to provide constant shaking to the algal cells to keep them in suspension and to ensure that they do not settle down on the bottom of the test vessel.
5. Study duration : The experimental phase of the study was lasted for a period of 72 hours.

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

50.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Effect were also observed at the concentration 50.12 mg/l.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

22.2 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

2) - Results with reference substance valid
- EC50: 0.77 mg/L (24 hours)

Reported statistics and error estimates:

EC50 was calculated using non linear regression by the software Prism 4.0

Validity criteria fulfilled:

not specified

Conclusions:

Based on the growth rate inhibition of green alga by the test chemical, the EC50 was determine to be in the concentration range of 22.2 mg/l to 50.12mg/L

Executive summary:

Data available for the structurally similar read across chemicals has been reviewed to determine the toxicity of aquatic algae of the test chemical .The studies are as mentioned below:

In the data for the lab report of the structurally similar read across substance ,the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test solution was prepared in aseptic condition. The test substance was prepared by adding 25.767 µl of test substance in 250 ml of BBM to get the final concentration of 103.07 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations 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 (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.After 72 hours of exposure of test organism with test chemical to various nominal test concentrations, EC50 was determine to be 50.26mg/L and 50.12mg/L graphically and through probit analysis. Based on the EC50, it can be concluded that the test chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

The above study was further supported by another structurally similar read across substance for the test chemical , toxicity of test material was evaluated for aquatic algae Desmodesmus subspicatus for 72 h , the EC50 was observed to be 22.2 mg/l. Based on the above effect concentration it can be considered that test material is toxic and can be classified as aquaticacute 3 as per CLP criteria.

Thus, based on the above summarized studies, test material and it’s structurally similar read across substance, it can be concluded that effect concentration value is in the range of 22.2 to 50.12mg/l. Thus, comparing this value with the criteria of CLP regulation, test material can be classified as aquatic acute 2 for algae and cyanobacteria

Description of key information

Toxicity to aquatic algae and cyanobacteria:

Data available for the structurally similar read across chemicals has been reviewed to determine the toxicity of aquatic algae of the test chemical .The studies are as mentioned below:

In the data for the lab report of the structurally similar read across substance ,the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test solution was prepared in aseptic condition. The test substance was prepared by adding 25.767 µl of test substance in 250 ml of BBM to get the final concentration of 103.07 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations 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 (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.After 72 hours of exposure of test organism with test chemical to various nominal test concentrations, EC50 was determine to be 50.26mg/L and 50.12mg/L graphically and through probit analysis. Based on the EC50, it can be concluded that the test chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

The above study was further supported by another structurally similar read across substance for the test chemical , toxicity of test material was evaluated for aquatic algae Desmodesmus subspicatus for 72 h , the EC50 was observed to be 22.2 mg/l. Based on the above effect concentration it can be considered that test material is toxic and can be classified as aquatic chronic 3 as per CLP criteria.

Thus, based on the above summarized studies, test material and it’s structurally similar read across substance, it can be concluded that effect concentration value is in the range of 22.2 to 50.12mg/l. Thus, comparing this value with the criteria of CLP regulation, test material can be classified as aquatic chronic 3 for algae and cyanobacteria

Key value for chemical safety assessment

EC50 for freshwater algae:

22.2 mg/L

Additional information

Toxicity to aquatic algae and cyanobacteria:

Data available for the structurally similar read across chemicals has been reviewed to determine the toxicity of aquatic algae of the test chemical .The studies are as mentioned below:

In the data for the lab report of the structurally similar read across substance ,the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test solution was prepared in aseptic condition. The test substance was prepared by adding 25.767 µl of test substance in 250 ml of BBM to get the final concentration of 103.07 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations 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 (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.After 72 hours of exposure of test organism with test chemical to various nominal test concentrations, EC50 was determine to be 50.26mg/L and 50.12mg/L graphically and through probit analysis. Based on the EC50, it can be concluded that the test chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

The above study was further supported by another structurally similar read across substance for the test chemical , toxicity of test material was evaluated for aquatic algae Desmodesmus subspicatus for 72 h , the EC50 was observed to be 22.2 mg/l. Based on the above effect concentration it can be considered that test material is toxic and can be classified as aquatic chronic 3 as per CLP criteria.

Thus, based on the above summarized studies, test material and it’s structurally similar read across substance, it can be concluded that effect concentration value is in the range of 22.2 to 50.12mg/l. Thus, comparing this value with the criteria of CLP regulation, test material can be classified as aquatic chronic 3 for algae and cyanobacteria