Diphenylacetonitrile

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 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 0 (vehicle control), 0.25, 0.5, 1, 2, and 4 mg/L
- Sampling method: Test item was weighed and dissolved in acetone, then transferred into a volumetric flask and made up to the required volume using natural water and coded as stock. Test solutions of the selected concentrations were prepared by dilution of the stock solution. The solutions were prepared in OECD medium. Volumetric flaks and measuring cylinder were used for solution preparation.
- Sample storage conditions before analysis: sample was analysed immediately after preperation

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 50 mg of test item was dissolve in 50 µL acetone and made up to 1000 mL in a volumetric flask using OECD medium (stock). 10 mL, 20 mL, 40ml, 80 mL, and 160 mL, from stock solution were transferred into 1L measuring cylinder and made up using OECD medium. Hence concentrations of 2, 4, 8, 16, 32 mg/L were achieved.
- Controls: OECD medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.1ml/L

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: ATCC
- Source (laboratory, culture collection): Initially procured from “American Type Culture Collection”, Chromachemie Laboratory Private Limited, later test system was sub-cultured in the test facility.
- Age of inoculum (at test initiation): 3-4 days before start of the test.
- Method of cultivation:The required volume of OECD growth medium was prepared as per OECD 201, test system was sub-cultured using OECD medium.

ACCLIMATION
- Culturing media and conditions (same as test or not):Yes
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.7 – 22.9 °C

pH:

7.6-8.0

Nominal and measured concentrations:

Nominal concentrations: control, solvent control, 2, 4, 8, 16, and 32 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Type (delete if not applicable): open / closed Sterilized, flasks were covered with cap
- Material, size, headspace, fill volume: Conical flasks were made of glass. Volume capacity 250 mL.
- Aeration: no aeration
- Initial cells density: 10000 cells/ ml
- Control end cells density: 10000 cells/ml
- No. of organisms per vessel: 10000 cells/mL
- No. of vessels per concentration (replicates): Main Study: 3 replicates/test item concentration
- No. of vessels per control (replicates): 6 replicates for control group during range finding and main study.
- No. of vessels per vehicle control (replicates):6 replicates for control group during range finding and main study.

GROWTH MEDIUM
- Standard medium used: yes Yes (OECD Medium as per OECD 201)


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:no
- Photoperiod:Continuous light for test system
- Light intensity and quality:Average: 6752-6784 Lux
- Salinity (for marine algae):
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cells were counted using Haemocytometer under illumination of the microscope at 24, 48, and 72 h after inoculation.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study Yes
- Test concentrations: 0 (control), 0 (vehicle control), 1.5, 3, 6, 12, 24 mg/L
- Results used to determine the conditions for the definitive study: Based on the results of range finding test following concentrations of 0 (control), 0 (vehicle control), 0.8, 1.6, 3.2, 6.4, 12.8 mg/L were selected for the main study as significant changes (inhibition algal growth rate) were observed in treatment groups during 72h test period.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 5.43 mg/L and 6.95 mg/L.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI 2.42 mg/L and 3.04 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes Yes (64.25, 62.58 times of the initial cell count in control and vehicle control, respectively)

Results with reference substance (positive control):

- Results with reference substance valid Yes
- EC50:
ErC50 (72h) = 0.620 mg/L (growth rate)
EyC50 (72h) = 0.334 mg/L (yield)

- Other:

Validity criteria fulfilled:

yes

Conclusions:

The effect of test chemical on fresh water algae was studied as per OECD 201 test guidelines, the 72-h EC50 of test item to alga on growth rate was reported to be 7.83mg/l

Executive summary:

This study was conducted as per OECD 201 (2011) to assess the acute toxicity effects of test chemical on the growth of green alga Pseudokirchneriella subcapitata(ATCC)following exposure of alga up to 72 h under static condition. Test system was initially procured from “American Type Culture Collection”, Chromachemie Laboratory Private Limited, later test system was sub-cultured (using OECD growth medium, OECD 201) in the test facility. 250 mL sterilized conical flasks covered with cap were used in the study. The test item was found to be soluble in acetone. Hence, acetone (concentration <0.1mL/L) with OECD growth medium was used as test medium. An inoculum culture in the test medium was prepared 3-4 days prior to test and culture conditions were maintained same as the test conditions.th test concentrations of 0 (control), 0 (vehicle control), 2, 4, 8, 16, 32 mg/L were selected for the main study(3 replicates/ concentration, 6 replicates for control groups)as significant changes (inhibition algal growth rate) were observed in treatment groups during 72h test period. The inhibition in growth rate was 0%, 0%, 2.1%, 26.4%, 52.2%, 80.3%, 92.7% were observed during 72h test period in the test concentrations of 0 (control), 0 (vehicle control), 2, 4, 8, 16, 32 mg/L. A reference standard (Potassium dichromate) was tested to ensure the authenticity of the test in the lab. And the inhibition of growth rate (E_rC₅₀-72h) and yield (E_yC₅₀-72h) were found to be 0.620 mg/L & 0.334 mg/L, respectively; and were found to be in acceptable range. The initial cell density was 10000 cells/mL.The algal cells in the control and vehicle control increased by >16 times of the initial cell count during the 72 -h exposure period, respectively. The mean coefficient of variation for section-by-section growth rate for the control cultures over the test period (0 -72 -h) was <35%. The coefficient of variation of average specific growth rate was <7%. Hence, fulfilling the all the validity criteria of the test. The analytical monitoring was done at 0 hour and 72 hours, and chemical was found to be maintained with in the acceptable range of 80 -120% of the nominal concentration, thus the results were reportedd in nominal concentraions. Continuous light having and average light intensity of 6752-6784 Lux, pH of 7.6-8.0, temperature 21.7-2329 °C along with a shaking speed of 110 RPM were provided and maintained for the test system throughout the test. 72h EC50 (growth rate and yield) values with 95% confidence limits were calculated by probit analysis. The cells were counted using Haemocytometer under illumination of the microscope at 24, 48, and 72 h after inoculation. 72-h EC₅₀of test item to alga on growth rate 7.83 mg/L, CI - 6.93 - 8.85mg/ l.

Thus, based on the EC50 value, test chemical can considered as toxic and hazardous to aquatic algae.Since the partition coefficient (log Kow) of the test chemical is ≤ 4 (i.e., reported as 2.79 to 3.2), test chemical can be considered as non-toxic to aquatic organisms and thuscan be considered to be 'Not classified’ as per CLP classification criteria.

Description of key information

This study was conducted as per OECD 201 (2011) to assess the acute toxicity effects of test chemical on the growth of green algaPseudokirchneriella subcapitata(ATCC)following exposure of alga up to 72 h under static condition. Test system was initially procured from “American Type Culture Collection”, Chromachemie Laboratory Private Limited, later test system was sub-cultured (using OECD growth medium, OECD 201) in the test facility. 250 mL sterilized conical flasks covered with cap were used in the study. The test item was found to be soluble in acetone. Hence, acetone (concentration <0.1mL/L) with OECD growth medium was used as test medium. An inoculum culture in the test medium was prepared 3-4 days prior to test and culture conditions were maintained same as the test conditions.th test concentrations of 0 (control), 0 (vehicle control), 2, 4, 8, 16, 32 mg/L were selected for the main study(3 replicates/ concentration, 6 replicates for control groups)as significant changes (inhibition algal growth rate) were observed in treatment groups during 72h test period. The inhibition in growth rate was 0%, 0%, 2.1%, 26.4%, 52.2%, 80.3%, 92.7% were observed during 72h test period in the test concentrations of 0 (control), 0 (vehicle control), 2, 4, 8, 16, 32 mg/L. A reference standard (Potassium dichromate) was tested to ensure the authenticity of the test in the lab. And the inhibition of growth rate (E_rC₅₀-72h) and yield (E_yC₅₀-72h) were found to be 0.620 mg/L & 0.334 mg/L, respectively; and were found to be in acceptable range. The initial cell density was 10000 cells/mL.The algal cells in the control and vehicle control increased by >16 times of the initial cell count during the 72 -h exposure period, respectively. The mean coefficient of variation for section-by-section growth rate for the control cultures over the test period (0 -72 -h) was <35%. The coefficient of variation of average specific growth rate was <7%. Hence, fulfilling the all the validity criteria of the test. The analytical monitoring was done at 0 hour and 72 hours, and chemical was found to be maintained with in the acceptable range of 80 -120% of the nominal concentration, thus the results were reportedd in nominal concentraions. Continuous light having and average light intensity of 6752-6784 Lux, pH of 7.6-8.0, temperature 21.7-2329 °C along with a shaking speed of 110 RPM were provided and maintained for the test system throughout the test. 72h EC50 (growth rate and yield) values with 95% confidence limits were calculated by probit analysis. The cells were counted using Haemocytometer under illumination of the microscope at 24, 48, and 72 h after inoculation. 72-h EC₅₀of test item to alga on growth rate 7.83 mg/L, CI - 6.93 - 8.85mg/ l.

Thus, based on the EC50 value, test chemical can considered as toxic and hazardous to aquatic algae. Based on the EC50 values chemical can be classified as aquatic category 2 as per CLP classification criteria

Key value for chemical safety assessment

EC50 for freshwater algae:

7.83 mg/L

Additional information

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

This study was conducted as per OECD 201 (2011) to assess the acute toxicity effects of test chemical on the growth of green algaPseudokirchneriella subcapitata(ATCC)following exposure of alga up to 72 h under static condition. Test system was initially procured from “American Type Culture Collection”, Chromachemie Laboratory Private Limited, later test system was sub-cultured (using OECD growth medium, OECD 201) in the test facility. 250 mL sterilized conical flasks covered with cap were used in the study. The test item was found to be soluble in acetone. Hence, acetone (concentration <0.1mL/L) with OECD growth medium was used as test medium. An inoculum culture in the test medium was prepared 3-4 days prior to test and culture conditions were maintained same as the test conditions.th test concentrations of 0 (control), 0 (vehicle control), 2, 4, 8, 16, 32 mg/L were selected for the main study(3 replicates/ concentration, 6 replicates for control groups)as significant changes (inhibition algal growth rate) were observed in treatment groups during 72h test period. The inhibition in growth rate was 0%, 0%, 2.1%, 26.4%, 52.2%, 80.3%, 92.7% were observed during 72h test period in the test concentrations of 0 (control), 0 (vehicle control), 2, 4, 8, 16, 32 mg/L. A reference standard (Potassium dichromate) was tested to ensure the authenticity of the test in the lab. And the inhibition of growth rate (E_rC₅₀-72h) and yield (E_yC₅₀-72h) were found to be 0.620 mg/L & 0.334 mg/L, respectively; and were found to be in acceptable range. The initial cell density was 10000 cells/mL.The algal cells in the control and vehicle control increased by >16 times of the initial cell count during the 72 -h exposure period, respectively. The mean coefficient of variation for section-by-section growth rate for the control cultures over the test period (0 -72 -h) was <35%. The coefficient of variation of average specific growth rate was <7%. Hence, fulfilling the all the validity criteria of the test. The analytical monitoring was done at 0 hour and 72 hours, and chemical was found to be maintained with in the acceptable range of 80 -120% of the nominal concentration, thus the results were reportedd in nominal concentraions. Continuous light having and average light intensity of 6752-6784 Lux, pH of 7.6-8.0, temperature 21.7-2329 °C along with a shaking speed of 110 RPM were provided and maintained for the test system throughout the test. 72h EC50 (growth rate and yield) values with 95% confidence limits were calculated by probit analysis. The cells were counted using Haemocytometer under illumination of the microscope at 24, 48, and 72 h after inoculation. 72-h EC₅₀of test item to alga on growth rate 7.83 mg/L, CI - 6.93 - 8.85mg/ l.

Thus, based on the EC50 value, test chemical can considered as toxic and hazardous to aquatic algae. Based on the EC50 values chemical can be classified as aquatic category 2 as per CLP classification criteria

 

In an experimental study, an acute test was conducted for 72 hrs for assessing the effect of test chemical on Desmodesmus subspicatus (Experimental study report, 2017). 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. 5x10(3) cells /ml was used as a test organism for the study. The stock solution 100 mg/ml was prepared by dissolving white powder in acetone. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Nominal test chemical conc. used for the study were 0, 0, 3, 6, 12, 24 and 50 mg/l, respectively. Study was performed using Desmodesmus subspicatus as a test organism in a static fresh water system. 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. 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. 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. 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 12.9 mg/l (95 % CI 11.5 - 14.6 mg/l).

 

In a supporting weight of evidence study from handbook (2008), short term toxicity to aquatic algae study was carried out for 96 hrs for assessing the effect of test chemical. The study was performed in a static fresh water system. Selenastrum capricornutum (Green algae) was used as test organism. On the basis of effect of the test chemical on growth rate of the test organism, the 96 hr EC50 value was determined to be 17 mg/l, respectively.

 

Thus, based on the EC50 value, test chemical can considered as toxic and hazardous to aquatic algae. Based on the EC50 values chemical can be classified as aquatic category 2 as per CLP classification criteria