2-furoic acid

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed using standard OECD test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test chemical was prepared by dissolving 50 mg of test substance in 250 ml of BBM to get the final concentration of 200 mg/l. The remaining test solutions were prepared by dilution from the above stock solution. All the final test solutions were prepared under aseptic condition. 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):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- 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

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 °C±2°C

Nominal and measured concentrations:

Nominal concentrations used for the study were 0, 6.25 mg/L,12.5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L and 200 mg/L. All the six concentration were in geometric series spaced by a factor of 2.

Details on test conditions:

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: 0, 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 22 ° 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):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: graphically and calculated from equation through probit analysis

Details on results:

The microscopic observations were also noted in each of the experimental flasks. All the cells appeared healthy, round and green throughout the test duration in the control and in the experimental flask also no significant changes were observed.

Reported statistics and error estimates:

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) was determined.

Table: Showing the values of average specific growth rate and percentage inhibition after an interval of 72 hours.

 

	Control		6.25 mg/l		12.5 mg/l		25 mg/l		50 mg/l		100 mg/l		200 mg/l
Average Specific Growth rate (μ )	R1	0.36	R1	0.35	R1	0.32	R1	0.30	R1	0.28	R1	0.24	R1	0.20
	R2	0.39	R2	0.33	R2	0.31	R2	0.29	R2	0.27	R2	0.22	R2	0.18
	R3	0.36
Mean of Avg. Specific growth rate			0.34		0.32		0.30		0.28		0.23		0.19
Percentage Inhibition (%I)	-		8.64		14.68		19.71		25.02		36.74		49.13

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth inhibition of green alga Chlorella vulgaris by the test chemical, the EC50 was determined to be >200 mg/l.

Executive summary:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Chlorella vulgaris (green algae) was used as a test organism for the study. Initial cell density of the culture was kept at 1 х 10000 cells/ml. Bold’s Basal Medium (BBM) composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium for the study. The test chemical was prepared by dissolving 50 mg of test substance in 250 ml of BBM to get the final concentration of 200 mg/l. The remaining test solutions were prepared by dilution from the above stock solution. All the final test solutions were prepared under aseptic condition. 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. Nominal test chemical concentration used for the study were 0, 6.25 mg/L,12.5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L and 200 mg/L. All the six concentration were in geometric series spaced by a factor of 2.Green algae were exposed to various nominal concentration of test chemical in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 22±2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 1500 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control (containing only the test medium) was also included in the test.The cultures were observed daily with the helpof an automated cell counter to verify a normal and healthy appearance of the algae cells 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 an automated cell counter.As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 16 within the 72 hr test period which corresponds to a specific growth rate of 0.92 per day, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% (i.e., reported as 24.22%) and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10% (i.e., reported as 5.09%), thus, fulfilling the validity of the criteria. In the control vessel, all cells appeared healthy,round and green throughout the study duration. The microscopic observations were also noted in each of the test vessel. All the cells appeared healthy, round and green throughout the study duration and no significant changes were observed up to the concentration of 200 mg/l. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs EC50 value was determined to be >200 mg/l.Thus, based on the EC50 value, chemical can be considered as non-toxic to aquatic algae and can be considered to be not classified as per CLP classification criteria.

 

Description of key information

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition.Chlorella vulgaris(green algae)was used as a test organism for the study. Initial cell density of the culture was kept at 1 х 10000 cells/ml. Bold’s Basal Medium (BBM) composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium for the study. The test chemical was prepared by dissolving 50 mg of test substance in 250 ml of BBM to get the final concentration of 200 mg/l. The remaining test solutions were prepared by dilution from the above stock solution. All the final test solutions were prepared under aseptic condition. 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. Nominal test chemical concentration used for the study were 0, 6.25 mg/L,12.5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L and 200 mg/L. All the six concentration were in geometric series spaced by a factor of 2.Green algae were exposed to various nominal concentration of test chemical in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 22±2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 1500 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control (containing only the test medium) was also included in the test.The cultures were observed daily with the helpof an automated cell counter to verify a normal and healthy appearance of the algae cells 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 an automated cell counter.As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 16 within the 72 hr test period which corresponds to a specific growth rate of 0.92 per day, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% (i.e., reported as 24.22%) and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10% (i.e., reported as 5.09%), thus, fulfilling the validity of the criteria. In the control vessel, all cells appeared healthy,round and green throughout the study duration. The microscopic observations were also noted in each of the test vessel. All the cells appeared healthy, round and green throughout the study duration and no significant changes were observed up to the concentration of 200 mg/l. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs EC50 value was determined to be >200 mg/l.Thus, based on the EC50 value, chemical can be considered as non-toxic to aquatic algae and can be considered to be not classified as per CLP classification criteria.

 

Key value for chemical safety assessment

EC50 for freshwater algae:

200 mg/L

Additional information

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

 

In an experimental study from study report, a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Chlorella vulgaris (green algae) was used as a test organism for the study. Initial cell density of the culture was kept at 1 х 10000 cells/ml. Bold’s Basal Medium (BBM) composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium for the study. The test chemical was prepared by dissolving 50 mg of test substance in 250 ml of BBM to get the final concentration of 200 mg/l. The remaining test solutions were prepared by dilution from the above stock solution. All the final test solutions were prepared under aseptic condition. 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. Nominal test chemical concentration used for the study were 0, 6.25 mg/L,12.5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L and 200 mg/L. All the six concentration were in geometric series spaced by a factor of 2.Green algae were exposed to various nominal concentration of test chemical in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 22±2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 1500 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control (containing only the test medium) was also included in the test. The cultures were observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells 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 an automated cell counter. As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 16 within the 72 hr test period which corresponds to a specific growth rate of 0.92 per day, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% (i.e., reported as 24.22%) and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10% (i.e., reported as 5.09%), thus, fulfilling the validity of the criteria. In the control vessel, all cells appeared healthy, round and green throughout the study duration. The microscopic observations were also noted in each of the test vessel. All the cells appeared healthy, round and green throughout the study duration and no significant changes were observed up to the concentration of 200 mg/l. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs EC50 value was determined to be >200 mg/l. Thus, based on the EC50 value, chemical can be considered as non-toxic to aquatic algae and can be considered to be not classified as per CLP classification criteria.

 

For the test chemical from authoritative database (2019), Toxicity to aquatic algae study was carried out for assessing the effect of test chemical. The study was performed following the principles of OECD Guideline 201 (Alga, Growth Inhibition Test) under static conditions. Based on the effect of test chemical on growth rate of the test algae, the 72 hr NOEC and EC50 value was determined to be 4.4 and > 58 mg/l, respectively.

 

On the basis of the above results, it can be concluded that the test chemical was considered to be non-toxic to aquatic algae and hence, considered to be 'not classified' as per the CLP classification criteria.