Salicylonitrile

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 September - 07 October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 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).
- Sampling method: Samples taken from test media (without algae) and at the end (containing algae).
- Sample storage conditions before analysis: Samples were deep-frozen immediately after sampling and stored at about -20˚C until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 100.05 mg of the test item completely in 1000 mL of test water using ultrasonic treatment for 15 minutes at room temperature. The test medium of the highest test concentration was diluted with test water to prepare the test media of the different test concentrations. The test media were prepared just before the start of the test.
- Controls: Reconstituted test water (OECD medium)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures, SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): 3 day old culture in exponential growth phase

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

24 mg/L as CaCO3

Test temperature:

22˚C

pH:

Test start range = 7.4 - 8.2
End test range = 7.6 - 9.6

Dissolved oxygen:

Not reported

Nominal and measured concentrations:

Nominal concentrations were: 0, 2.2, 4.6, 10, 22, 46 and 100 mg/L. Measured concentrations at day 0 were: 0, 2.32, 4.97, 10.8, 23.8, 48.7 and 99.8 mg/L. Measured concentrations at day 4 were: 0, 2.33, 4.82, 10.7, 23.4, 47.8 and 99.8 mg/L. The measured concentrations of the test substance in the test media for 2.2 to 100 mg/L were between 100 and 108% of the nominal values at the start of the test, and between 100 and 107% at the end of the test. Thus the correct dosing was confirmed and the test item is stable; the results are based on nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flask
- Type: closed, covered with a glass dish
- Material, size, headspace, fill volume: Fill volume = 15 mL
- Initial cells density: 5000 cells/mL

- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (OECD medium)
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS

- Photoperiod: Constant illumination
- Light intensity and quality: 4510 - 4880 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Fluorescence

TEST CONCENTRATIONS

- Test concentrations: 0, 2.2, 4.6, 10, 22, 46 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (tested twice per year)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 9.2 - 20

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 55 - 73

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 7.5 - 18

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 22 - 38

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 66 - 72

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 33 - 38

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities were observed

72 h NOECs (biomass, growth rate and yield) and the 96 h NOEC for biomass could not be determined in this study.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 72 hour growth rate (March 2010) 0.94 mg/L
- Other: Results from Harlan Laboratories study C86922

Reported statistics and error estimates:

72 and 96 hour LOEC and NOEC values were determined using Williams' t-test or Welch t-test. 72 and 96 hour EC50 values were calculated by probit analysis.

The test item had a significant inhibitory effect on the AUC of the algae after the test periods of 72 and 96 hours at the concentration of 2.2 mg/L and at all higher test concentrations (results of Williams and Welch t-tests, one-sided,α= 0.05). Thus, this concentration was determined to be the 72 and 96-hour LOEC.

The 72 and 96-hour NOEC could not be determined since at the lowest nominal test concentration of 2.2 mg/L the AUC of the algae was significantly lower than in the control. The 72- and 96-hour EC50 values for the inhibition of AUC, average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis.

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 46 mg/L and the algal cells in the control. The shape and size of the algal cells was obviously not affected by the test item up to at least this concentration.

In the control the biomass increased by a factor of 121 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control during 72 and 96 hours was 4.4 and 32%, respectively.

According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 and 96 hours was 0.7 and 0.5%, respectively. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

Validity criteria fulfilled:

yes

Conclusions:

In a guideline, 96-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the substance exhibited EC50 values of 29, 69 and 33 mg/L based on biomass, growth rate and yield, respectively. The 96 hour NOEC was 22 mg/L for growth rate and yield, but could not be determined for biomass. The 96 hour LOEC was 2.2 mg/L for biomass and 46 mg/L for growth rate and yield. All results are based on nominal concentrations.

Executive summary:

In a GLP-compliant, guideline 96-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the substance exhibited EC50 values of 29, 69 and 33 mg/L based on biomass, growth rate and yield, respectively. The 96 hour NOEC was 22 mg/L for growth rate and yield (the biomass NOEC could not be determined). The 96 hour LOEC was 2.2 mg/L for biomass and 46 mg/L for growth rate and yield. All results are based on nominal concentrations.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Apr - 7 May 2010

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:

yes

Remarks:

No analytical monitoring

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

yes

Remarks:

temp between 20 - 22°C - Guideline requires 24 ± 2°C

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

No analytical monitoring

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test medium at the highest nominal concentration, 100 mg/L, was prepared by dissolving 40.04 mg of test item in 400 mL test water with 15 minutes ultrasonic treatment and 15 minutes intense stirring. The test series was prepared by diluting samples of this solution with appropriate volumes of test water
- Controls: Test water without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Insititute for Plant Physiology, University of Göttingen / Germany)
- Age of inoculum (at test initiation): 3 days. Algae were in the exponential growth phase
- Method of cultivation: Standardized conditions according to guidelines.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: None reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

24 mg/L as CaCO3

Test temperature:

21 °C

pH:

At test start: 7.3 - 8.0
At test end: 7.5 - 9.0

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

0 (control), 2.2, 4.6, 10, 22, 46 and 100 mg test item/L. All values nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: Flasks
- Type (delete if not applicable): Erlenmeyer flasks covered with glass dishes
- Material, size, headspace, fill volume: 50-mL Erlenmeyer flasks containing 15 mL of test solution
- Aeration: Stirred by magnetic stirrers
- Initial cells density: 5000 cells/mL
- Control end cells density: Not calculated
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconsituted test water according to OECD guideline 201
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuous
- Light intensity and quality: Mean: 4200, Range: 4010 - 4430 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Electronic particle counter
- Other: Algae in the control and 46 mg/L test solution were compared microscopically at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study: Yes
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, tested twice during year

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

area under the growth curve

Remarks on result:

other: 95% CL: 29 - 34 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

area under the growth curve

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 60 - 70 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% CL: 28 - 32 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

area under the growth curve

Remarks on result:

other: 95% CL: 30 - 33 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

area under the growth curve

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 60 - 67 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% CL: 30 - 34 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Control and 46 mg/L algae compared. No differences found.
- Unusual cell shape: No
- Colour differences: None reported
- Flocculation: None reported
- Adherence to test vessels: None reported
- Aggregation of algal cells: None reported
- Any stimulation of growth found in any treatment: No significant effects
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid?: Yes, tested March 2010
- EC50: 0.94 mg Potassium dichromate/L (72 hours, growth rate)
- Other: Sensitivity of test system demonstrated, internal historical range 72-h EC50 0.71 - 1.7 mg/L

Reported statistics and error estimates:

The EC50 values and their 95% confidence limits were calculated by Probit analysis.
The test concentration results were compared to the control values by William's tests.

Validity criteria fulfilled:

yes

Remarks:

Control biomass increased by a factor of 114 over 72 hours; mean coefficient of variation in control daily growth rates was 4.0 and 20.6% during 72 and 96 hours, respectively, and coefficient of variation of average specific growth rates in control replic

Conclusions:

In a 96-hour algal growth inhibition test test with Pseudokirchneriella subcapitata the substance exhibited EC50 values of 31, 63 and 32 mg/L based on biomass, growth rate and yield respectively. The 96 hour NOEC is 10 mg/L for all assessment parameters.

Executive summary:

In a GLP-compliant, guideline 96-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the substance exhibited EC50 values of 31, 63 and 32 mg/L based on biomass, growth rate and yield, respectively. The 96 hour NOEC was 10 mg/L for all assessment parameters. All results are based on nominal concentrations.

Description of key information

ErC50=63mg/L (95% C.I.=60-67 mg/L), NOErC=10mg/L, 96h, Pseudokirchneriella subcapitata, OECD 201, Höger 2010
ErC50=69mg/L (95% C.I.=66-72 mg/L), NOErC=22mg/L, 96h, Pseudokirchneriella subcapitata, OECD 201, Liedtke 2010

Key value for chemical safety assessment

EC50 for freshwater algae:

63 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

The influence of the test substance on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 96-hour static test under GLP and according to OECD Guideline 201 (2006), by Höger (2010). The nominal concentrations of the test item of 2.2, 4.6, 10, 22, 46 and 100 mg/L were tested in parallel with a control. No analytical measurements were performed in the test. The 96h ErC50 was found to be 63 mg/L (60-67 mg/L, CI=95%), and a NOEC(growth rate)=10 mg/L determined, based on nominal concentrations.

 

In a further study, the influence of the test substance on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 96-hour static test under GLP and according to OECD Guideline 201 (2006), by Liedtke (2010). The nominal concentrations of the test item of 2.2, 4.6, 10, 22, 46 and 100 mg/L were tested in parallel with a control. The measured concentrations of the test substance in the test media for 2.2 to 100 mg/L were between 100 and 108% of the nominal values at the start of the test, and between 100 and 107% at the end of the test. Thus the correct dosing, and stability of the substance in the test media, was confirmed.The 96h ErC50 was found to be 69mg/L (66-72mg/L, CI=95%), and a NOEC(growth rate)=22 mg/L determined, based on nominal concentrations.

The two available algae studies are in excellent agreement for growth rate and yield at 96-hours. Some parameters in the study by Liedtke (2010) could not be determined at 72-hours, and the 96-hour AUC could not be determined.

 

The chemical structure is not expected to be hydrolytically unstable, nor is there experimental evidence to suggest this. Stability of salicylonitrile in the test media was confirmed by Liedtke with analytical monitoring, and considering the good agreement between the effect concentrations in the two studies, it can be concluded that the results determined by Höger are fully reliable, despite the lack of measured concentrations. Because the latter results are slightly lower, and more conservative, these are the values taken forward for risk assessment and classification.

 

The available studies are considered to be reliable, adequate, and conclusive for risk assessment and classification.