2-nitro-4-(trifluoromethyl)benzonitrile

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 – 27 Apr 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

GLP compliance:

yes

Remarks:

The test was performed in compliance with the requirements of the Principles of Good Laboratory Practices (GB/T 22278-2008).

Test material

Test material information

Constituent 1

Reference substance name:

2-nitro-4-(trifluoromethyl)benzonitrile

EC Number:

212-298-1

EC Name:

2-nitro-4-(trifluoromethyl)benzonitrile

Cas Number:

778-94-9

Molecular formula:

C8H3F3N2O2

IUPAC Name:

2-nitro-4-(trifluoromethyl)benzonitrile

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.0 (Control), 0.0625 mg/L, 0.125 mg/L, 0.25 mg/L, 0.50 mg/L, and 1.00 mg/L
- Sampling method: At 0 h, 24 h, 48 h, and 72 h the test solution was collected and changes in test substance concentration were analysed. 30 mL samples were taken from the blank control group and the 0.0625 mg/L and 0.125 mg/L test groups. 10 mL samples were taken from the 0.25 mg/L, 0.50 mg/L, and 1.00 mg/L test groups.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Method for preparing the test substance stock solution (100 mg/L) for the actual test: 0.0101 g test substance was weighed, and sterilised, then using AAP this was diluted to 100 mL after being subjected to ultrasound for 20 minutes. Secondary stock solution (10 mg/L): 10 mL of 100 mg/L stock solution was aspirated, and sterilised AAP used to dilute to 100 mL, yielding a 10 mg/L stock solution. The stock solution was placed in 150 mL Erlenmeyer flasks according to Table 1 in “Any other information on materials and methods incl. tables”, sterilised AAP culture medium was added to dilute to the different concentrations of test solution.
- Controls: Negative control consisting of test medium

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Batch number: A20190419P
- Source: Freshwater Algae Culture Collection of the Chinese Academy of Sciences
- Method of cultivation: The algae strain was continuously cultured at this laboratory using AAP culture medium, transferred once weekly, and inoculation quantity was 1% of the algal cell concentration prior to inoculation.
- Culturing before testing: Prior to starting the test, 1.00 mL of Pseudokirchneriella subcapitata algal solution was inoculated into 100 mL of sterilised culture medium, placed in a shaking culture box at a temperature of 23.4°C, illuminated at 5000 Lux (± 15%) and pre-cultured for 4 to 5 days at a shaking speed of 100 r/min.
- Other: Prior to the test, algal growth conditions in the stock solution were checked, and once it was discovered that algal cell growth was good, without cellular abnormalities, bacteria, other algal contamination, or anomalous phenomena, said algal strain met the test requirements.

ACCLIMATION
- Culturing media and conditions: Same conditions, but cultured in 100 mL (preculture) instead of 70 mL (test)
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23.0 - 23.6 °C

pH:

During the test: 7.48 - 7.98 (Blank control), 7.21 - 7.96 (treatments)

Nominal and measured concentrations:

Nominal concentrations: 0.0 (Control), 0.0625, 0.125, 0.25, 0.50 and 1.00 mg/L
Mean measured concentrations: < LOD/LOQ, 0.0597, 0.124, 0.244, 0.455 and 0.950 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL Erlenmeyer flasks
- Material, size, headspace, fill volume: 70 mL of test substance solution and algal cells
- Initial cells density: 1.76 x10^4 cells/mL
- Control end cells density: 64.2 – 70.2 x10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per positive control (replicates): 3

GROWTH MEDIUM
- Standard medium used: Yes, AAP culture medium was used.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water was used to prepare test medium
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH was measured at the beginning (0 h) and end of test (72 h)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous
- Light intensity and quality: 5617-5700 Lux, uniform cool white light
- Shaking rate: 100 r/min

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell density was measured at 0, 24, 48, and 72 hours using a microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 0.0 (Control), 1.00 mg/L, 10.0 mg/L, and 100 mg/L
- Results used to determine the conditions for the definitive study: 91.0% growth inhibition at 1.00 mg/L after 72 h, 107% growth inhibition after 72 h at 10.0 mg/L and 100 mg/L.

CULTURING APPARATUS
- Details on culturing apparatus used: Illuminated shaker (JM-C, Jiangsu Jiamei Instrument Manufacturing Co., Ltd.); Erlenmeyer flasks (150 mL, Tianbo Instruments Factory).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate was used as positive control and tested at least once a year, to verify that the test conditions satisfied the test requirements and no significant changes had occurred.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.38 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.247 mg/L

Nominal / measured:

meas. (initial)

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): During the actual test, algal cell morphology in the blank control group was normal; with the exception of reduced algal cell quantity in the test groups, no other abnormal phenomena were observed.
- Other: The results indicate that, under the conditions of this test, at the end of the test (72 h) the test substance concentration was 94.3-99.2% that of the initial concentration (0 h) and the change in concentration was < 20% that of the initial concentration. Therefore, test system concentration was stable in the test process, and the test results were expressed using initial actual measured concentration.

Results with reference substance (positive control):

- EC50 (72 h): 1.23 mg/L (nominal) obtained in last reference compound test on 15 to 18 Jan 2019.

Reported statistics and error estimates:

The trimmed Spearman-Karber method (version 1.5, USEPA) was used to calculate ErC50 and 95% confidence interval.
The Dunnett’s test (Dunnett’s tests, ANOVA, SPSS 19.0) was used to analyse algal growth rates in the test groups and the control group, and determine the lowest observed effect concentration (LOEC) (p < 0.05) and the no observed effect concentration (NOEC).

Any other information on results incl. tables

Table 1: Results from Growth Inhibition Test of NTBN on Pseudokirchneriella Subcapitata

Prepared concentration^1	24 h		48 h		72 h
	Cell conc.^2	Inhibition rate (%)	Cell conc.^2	Inhibition rate (%)	Cell conc.^2	Inhibition rate (%)
blank control^2	4.66	-	17.1	-	64.2	-
	4.76		19.0		67.8
	4.76		18.4		70.2
0.0625 mg/L	3.86	7.07	17.0	3.15	64.4	-0.0844
	4.76		17.7		73.0
	4.66		16.0		65.6
0.125 mg/L	4.46	13.9	16.9	9.31	78.5	0.928
	3.96		13.1		55.3
	3.96		14.1		63.6
0.25 mg/L	3.66	25.0	13.1	14.1	62.6	3.21
	3.56		13.1		55.6
	3.86		13.0		61.8
0.50 mg/L	2.36	70.3	2.76	79.2	3.66	80.3
	2.36		2.96		4.06
	2.36		2.86		3.16
1.00 mg/L	14.96	87.4	2.16	90.6	2.16	94.8
	2.06		2.26		2.16
	1.96		2.16		2.06
ErC50 (mg/L) (based on initial actual measured concentration value)	0.36		0.36		0.38
95% confidence interval (mg/L)	0.34 – 0.37		0.35 – 0.37		0.38 – 0.39

¹Algal cell initial concentration at 0 h was 1.76 x 10⁴cells/mL.

²(x10⁴cells/mL)

 

  Table 2: Measurement Results for NTBN Stability in Test Medium

Prepared concentration (mg/L WAFs)	Actual measured concentration (mg/L)					72 h actual measured concentration/ 0 h actual measured concentration (%)
	0 h	24 h	48 h	72 h	Mean value
Blank control	ND^1	ND	ND	ND	-	-
0.0625	0.0613	0.0589	0.0602	0.0582	0.0597	94.9
0.125	0.124	0.126	0.123	0.123	0.124	99.2
0.25	0.247	0.249	0.240	0.240	0.244	97.2
0.50	0.471	0.448	0.451	0.450	0.455	95.5
1.00	0.992	0.930	0.942	0.935	0.950	94.3

¹: not detected.

Table 3: Validity criteria for OECD 201 (2006)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	During the 72 h test period, algal cells in the blank control group presented exponential growth and concentration increased by a factor of 38.	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	For each test stage over the 72 h (e.g., 0-1 days, 1-2 days, 2-3 days), mean coefficient of variation of the daily specific growth rate was 16%.	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	For each parallel in the control group, mean coefficient of variation of the daily specific growth rate was 1.24%.	yes

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See Table 1 in "Any other information on results incl. tables".