2-nitro-4-(trifluoromethyl)benzonitrile

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Mar – 21 Jun 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 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

2010

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:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: During the test, certain quantities of test substance were weighed directly and added to the test bottles.
- Controls: Yes. Reference compound 3,5-dichlorophenol stock solution (1000 mg/L): 0.1002 g 3,5-dichlorophenol was weighed, diluted with deionised water to approximately 100 mL, yielding 3,5-dichlorophenol solution with a pH adjusted to 7.82 and a concentration of 1000 mg/L (Positive control). Inoculum blank control without test substance.
- Test substance concentrations: 0.0 (blank control), 10, 20, 40, 80, and 160 mg/L
- Reference compound concentrations: 3, 10, and 30 mg/L

Test organisms

Test organisms (species):

activated sludge

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Nanjing Tiebei Sewage Treatment Plant
- Method of cultivation: The sewage sludge was washed with deionised water, centrifuged at high-speed for 10 minutes at 5000 r/min, and the supernatant poured out. The operation was repeated three times, a small quantity of washed sludge was weighed and dried, and dry sludge content calculated to be 6.79%. Based on sludge dry-weight content, 265.30 g of centrifuged wet sludge (sludge dry weight 18.0 g) was added to 300 mL synthetic sewage, and deionised water used to dilute to 6 L, yielding a 3.00 g/L activated sludge suspension, and the wet sludge stirred for 2 minutes to homogenise.
- Preparation of inoculum for exposure: Before the test started, the activated sludge suspension was aerated for 16 h at (20 ± 2) °C; the measured pH value was 7.41.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

20 ± 2 °C

pH:

7.62 - 7.96

Dissolved oxygen:

0.07 - 7.97

Details on test conditions:

TEST SYSTEM
- Aeration: Aerated with continuous stirring. Clean, de-oiled air.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The respiration rate of the test solution was measured every 15 minutes

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.0 (blank control), 10, 100, and 1000 mg/L
- Results used to determine the conditions for the definitive study: Dissolved oxygen before and after the abiotic control test was 6.71 mg/L and 6.71 mg/L respectively; there were no pronounced abiotic aerobic effects. Test substance 3 h-EC50 = 103 mg/L, the 95% confidence interval was 66.9 mg/L - 159 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

35.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Total respiration rate for the blank control was 46.1 mgL-1 h-1 - 48.1 mgL-1 h-1, which converts to an activated sludge (1.5 g/L) respiration rate of 30.8 mgg-1 h-1 - 32.0 mgg-1 h-1 (> 20 mgg-1 h-1), and the coefficient of variation was 1.49% (< 30%).
The respiration rates for inoculum blank controls without ATU and with ATU were 41.1 mgL-1 h-1 and 39.6 mgL-1 h-1; inhibition rate for nitrification respiration was 3.57% (< 5%), indicating that nitrification effects were not significant, and that this could be used in the actual test.

Results with reference substance (positive control):

The 3 h total respiration inhibition EC50 value for the reference compound 3,5-dichlorophenol was 13.1 mg/L, the 95% confidence interval was 11.4 mg/L - 15.0 mg/L (between 2-25 mg/L).

Any other information on results incl. tables

 Table 1: Total Respiration Inhibition Test Results

Treatment	Conc. (mg/L)	Dissolved oxygen (mg/L)		Time (s)	Respiration rate (mgL^-1h^-1)	Inhibition rate (%)
		Upper limit	Lower limit
1 Blank control FB1	0	7.25	0.07	540	47.9	—
2 Test substance FT1-1	10.1	7.35	0.37	540	46.5	1.62
3 Test substance FT1-2	10.0	7.24	0.24	540	46.7	1.34
4 Test substance FT1-3	10.1	7.53	0.63	540	46.0	2.75
5 Test substance FT1-4	10.1	7.14	0.27	540	45.8	3.17
6 Test substance FT1-5	10.3	7.26	0.33	540	46.2	2.33
7 Test substance FT2-1	20.1	7.97	1.57	540	42.7	9.80
8 Test substance FT2-2	19.9	7.55	1.66	540	39.3	17.0
9 Test substance FT2-3	19.9	7.31	1.15	540	41.1	13.2
10 Test substance FT2-4	19.9	7.23	1.03	540	41.3	12.6
11 Test substance FT2-5	20.1	7.37	1.06	540	42.1	11.1
12 Test substance FT3-1	40.0	7.18	5.68	540	10.0	78.9
13 Test substance FT3-2	40.2	7.86	6.69	540	7.80	83.5
14 Test substance FT3-3	40.2	7.51	6.30	540	8.07	82.9
15 Test substance FT3-4	39.8	7.46	6.03	540	9.53	79.8
16 Test substance FT3-5	40.0	7.38	5.97	540	9.40	80.1
17 Reference control FR4	3.00	7.37	0.55	540	45.5	3.88
18 Reference control FR5	10.0	7.28	2.79	540	29.9	36.7
19 Reference control FR6	30.0	7.57	6.43	600	6.84	85.5
20 Blank control FB2	0	7.12	0.09	540	46.9	—
21 Blank control FB3	0	7.56	0.48	540	47.2	—
22 Blank control FB4	0	7.88	0.77	540	47.4	—
23 Test substance FT4-1	80.0	7.20	6.61	600	3.54	92.5
24 Test substance FT4-2	80.0	7.27	6.12	600	6.90	85.4
25 Test substance FT4-3	80.2	7.21	6.60	600	3.66	92.3
26 Test substance FT4-4	80.0	7.28	6.64	600	3.84	91.9
27 Test substance FT4-5	80.2	7.22	6.55	600	4.02	91.5
28 Test substance FT5-1	160	7.28	7.00	600	1.68	96.4
29 Test substance FT5-2	160	7.89	7.41	600	2.88	93.9
30 Test substance FT5-3	160	7.55	7.32	600	1.38	97.1
31 Test substance FT5-4	160	7.19	6.98	600	1.26	97.3
32 Test substance FT5-5	160	7.33	7.14	600	1.14	97.6
33 Reference control FR4	3.0	7.66	0.70	540	46.4	1.90
34 Reference control FR5	10	7.38	2.75	540	30.9	34.7
35 Reference control FR6	30	7.41	6.46	600	5.70	87.9
36 Blank control FB5	0	7.64	0.72	540	46.1	—
37 Blank control FB6	0	7.82	0.61	540	48.1	—
Test substance EC50	35.6 (mg/L)	Test substance EC50 95% confidence interval (mg/L)			31.9 – 39.7 mg/L
Reference compound EC50	13.1 (mg/L)	Reference compound EC50 95% confidence interval (mg/L)			11.4 – 15.0 mg/L

Notes: Coefficient of variation for the respiration rate of the inoculum blank control was 1.49% (within 30%), total respiration rate of the blank control was 46.1mgg^-1h^-1- 48.1mgL^-1h^-1, which converts to an activated sludge (1.5 g/L) respiration rate of 30.8mgg^-1h^-1- 32.0 mgg^-1h^-1.

 

Table 2: Validity criteria for OECD 209

Criterion from the guideline	Outcome	Validity criterion fulfilled
The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.	Total respiration rate for the blank control was 30.8 mg*g^-1*h^-1- 32.0 mg*g^-1*h^-1.	yes
The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.	The coefficient of variation of oxygen uptake rate in control replicates was 1.49% (< 30%).	yes

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See Table 2 in "Any other information on results incl. tables".