4-mesyl-2-nitrotoluene

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

Study period:

18 May 1999 to 21 May 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from the exposure concentrations and control.
- Sampling method: Samples were taken from the test medium at the start using excess remaining after filling the test vessels. At the end of the test each blank solution (without algal inoculum) was sampled and analysed in the same manner.
- Sample storage conditions before analysis: Not reported

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 2000 ml volume of the highest nominal concentration (180 mg/L) was prepared by addition of the test substance to sterile culture medium. Aliquots of this were added to sterile culture medium to give the test concentrations.
- Controls: Yes, a blank control of culture medium only
- 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:

- Common name: green algae
- Strain: ATCC 226622
- Source (laboratory, culture collection): Lab culture maintained under axenic conditions
- Age of inoculum (at test initiation): 4 days (exponential growth phase)
- Method of cultivation: Grown in test medium

ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: Not reported.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

24 ± 1 °C measured daily

pH:

At start of test: 7.2 - 7.4
At end of test: 7.5 - 9.1

Dissolved oxygen:

Not reported

Nominal and measured concentrations:

See Table 1. Measured concentrations ranged from 100 - 110 % nominal, so results are reported as nominal concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask
- Type (delete if not applicable): Closed (polyuethane foam bungs)
- Material, size, headspace, fill volume: Borosilicate galss, 250 mL capacity, 100 mL of test solution
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter): Static, none used
- Renewal rate of test solution (frequency/flow rate): Static test
- Initial cells density: 10000 cells/mL
Control end cells density: 2350000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, as per USEPA Protocol EPA-600/9-78-018

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Distilled water
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/Mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: Not reported

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: None
- Photoperiod: Constant illumination
- Light intensity and quality: 7560 lux (measured once)
- Salinity (for marine algae): Not applicable
- Shaking: 160 rpm, orbital shaking.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter counter model ZM
- Chlorophyll measurement: Not reported

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.1
- Test concentrations: 1.0, 2.1, 4.5, 9.5, 20, 41, 86, 180 mg/L

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 11-21 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 56-75 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.5 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
- Observation of abnormalities (for algal test):
- Unusual cell shape: Not reported
- Colour differences: Not reported
- Flocculation: Not reported
- Adherence to test vessels: Not reported
- Aggregation of algal cells: Not reported
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: No

Reported statistics and error estimates:

The EC50s and their 95% confidence intervals were calculated by a Weibull model. For the determination of the NOEC the results from the test concentrations were compared to the control values by Dunnett's tests (P = 0.05).

Table 2: Mean growth rates over the test period

Nominal concentrations (mg/L)	Mean growth rate (0 - 3 days)	Percentage of culture medium control
Control	1.794	-
1	1.774	99
2.1	1.77	99
4.5	1.703*	95
9.5	1.658*	92
20	1.425*	79
41	1.147*	64
86	0.766*	43
180	0.235*	13

*mean value significantly lower than in control

Table 3: Mean areas under the growth curve

Nominal concentrations (mg/L)	Mean area under the growth curve (0 - 3 days)	Percentage of culture medium control
Control	153.3	-
1	148.6	97
2.1	145.7	95
4.5	121.0*	79
9.5	109.9*	72
20	60.4*	39
41	28.2*	18
86	9.9*	6
180	1.7*	1

*mean value significantly lower than in control

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal concentrations, the 72-hour ErC50 for the test substance to pseudokirchnerella subcapitata (selenastrum capricornutum) was 66 mg/L and the EbC50 was 16 mg/L. The 72 h NOECs were 2.1 mg/L (biomass and growth rate).

Executive summary:

The toxicity of the test substance to the green alga selenastrum capricornutum was determined. In accordance with the guidelines, algae were exposed to nominal concentrations of 1.0, 2.1, 4.5, 9.5, 20, 41, 86 and 180 mg/L. Based on nominal concentrations, the 72-hour ErC50 was 66 mg/L and the EbC50 was 16 mg/L. The 72 h NOECs were 2.1 mg/L (biomass and growth rate).

Description of key information

ErC50=66 mg/L, NOEC=2.1 mg/L (growth rate), 72h, Pseudokirchnerella subcapitata, OECD 201, Smyth 1999

Key value for chemical safety assessment

EC50 for freshwater algae:

66 mg/L

EC10 or NOEC for freshwater algae:

2.1 mg/L

Additional information

The toxicity of the test substance to the green alga (Pseudokirchnerella subcapitata) was determined. In accordance with the guidelines, algae were exposed to nominal concentrations of 1.0, 2.1, 4.5, 9.5, 20, 41, 86 and 180 mg/L. Based on nominal concentrations, the 72-hour ErC50 was 66 mg/L and the EbC50 was 16 mg/L. The 72 h NOECs were 2.1 mg/L (biomass and growth rate). The study is considered to be reliable and adequate for the purposes of risk assessment, classification and labelling.