2-hydroxy-4-(trifluoromethyl)benzoic acid

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 16th, 2019 to April 11th, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Remarks:

test item directly weighed into the test vessels.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test vessels containing distilled water.
- Eluate: distilled water
- Controls: blank control, abiotic control (only for range finding test), positive control (reference substance).
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Activated sludge was collected from Sewage Treatment Plant (SIT), Chandana Complex, Tumkur – 572103, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was collected, the coarse particles was were removed by settling for a short period for about 15 minutes, and decanting the upper layer of finer solids. The sludge was washed and centrifuged for a period of 10 minutes to produce a clear supernatant and pellet of sewage solids. The supernatant liquid was discarded, and the sludge re-suspended in distilled water with shaking and the wash-water was removed by re-centrifuging and discarding again. The washing and centrifuging process was repeated twice. The dry mass of a known volume of the re-suspended sludge was determined and the sludge was concentrated by diluted further in distilled water to obtain the required sludge stock solids concentration of about 3 g/L. The activated sludge was continuously aerated at the test temperature until usage.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2°C

pH:

The pH in all test vessels was adjusted at 7.5 ± 0.5 (7 - 8).

Dissolved oxygen:

60-70% saturation (> 5 mg/L at 20ºC)

Nominal and measured concentrations:

- range-finding test: 0(control), 10, 100, 1000 mg/L test item (nominal).
- main test: 0 (control), 100, 178, 316, 563, 1000 mg/L test item (nominal).

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000mL glass beakers
- Type: open
- Material, size, headspace, fill volume: glass, size 1000mL, fill volume 500 mL.
- Aeration: yes. Before the start of the test, the aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (> 5 mg/L) and to maintain the sludge flocs in suspension (approx. 0.6 L/min).
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 0 (no oxygen uptake observed in the range-finding test).
- Sludge concentration (weight of dry solids per volume): 3.0 g/L
- Weight of dry solids per volume of reaction mixture per unit of time: 1.5 g/L
- Nutrients provided for bacteria: 50 mL of synthetic medium per litre of activated sludge.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: distilled water

OTHER TEST CONDITIONS
- Adjustment of pH: yes.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Oxygen consumption was measured over a period of 10 min after 3 h incubation of the test vessels. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls. The medium temperature was recorded continuously in temperature control vessels.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.78
- Range finding study : yes. The final test was performed based on the result of a preceding combined limit/range-finding test.
- Test concentrations: 10, 100 and 1000 mg/L in the range finding test; 100, 178, 316, 563, and 1000 mg/L in the final test.
- Results used to determine the conditions for the definitive study: The combined limit/range-finding showed no inhibition, 24% and 91% inhibition of the respiration rate at a concentration of 10, 100 and 1000 mg/L, respectively. Therefore, the expected EC50 was between 100 and 1000 mg/L. Also, there was a significant oxygen production from the abiotic processes. The oxygen production was 17% of the mean respiration rate of the control. Therefore it was decided to perform the final tests with an addition of an abiotic control of all concentrations.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (Sigma Aldrich)

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

178 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

396.37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: (95% CL: 336.11-467.42 mg/L)

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

250.27 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: (95% CL: 182.93-342.40 mg/L)

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

293.07 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: (95% CL: 229.56-374.15 mg/L)

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

536.07 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: (95% CL: 429.63-668.89 mg/L)

Details on results:

- No statistically significant inhibition of the respiration rate of the sludge was recorded at 178 mg/L test item. At higher concentrations the inhibitory effect of the test item on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 20% inhibition at 316 mg/L to 99% at 1000 mg/L.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
- Effect concentrations exceeding solubility of substance in test medium: yes.
- Adsorption (e.g. of test material to the walls of the test container): no.
- Blank controls oxygen uptake rate: The mean control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (29 mg oxygen per one gram of activated sludge in the final test).
- Coefficient of variation of oxygen uptake rate in control replicates: The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test (18%).

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (3.2 mg/L in the final test).

Reported statistics and error estimates:

Calculation of the ECx value was based on probit analysis using linear maximum likelihood regression, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item. Calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 2. Summary of test results.

Details	EC-values	Confidence Interval P = 95 [%]
		Lower Limit	Upper Limit
NOEC*	178 mg/L	-	-
EC10	250.27 mg/L	182.93 mg/L	342.40 mg/L
EC20	293.07 mg/L	229.56 mg/L	374.15 mg/L
EC50	396.37 mg/L	336.11 mg/L	467.42 mg/L
EC80	536.07 mg/L	429.63 mg/L	668.89 mg/L

Table 3. Percentage of inhibition data after 3 hours for the test item.

The mean total respiration inhibition for the test item with five replicates at the nominal concentrations of 100 mg/L, 178 mg/L, 316 mg/L, 563 mg/L, and 1000  mg/L were found to be  -13.85%, -7.78%, 20.43%, 91.47% and 99.14%, respectively.

Sample Code	Respiration Rate (R) mg / L × h	Specific Respiration Rate (Rs) mg O2/ g × h		Mean Specific Respiration Rate (Rs) mg O2/ g × h	Inhibition Rate (%)		Mean Inhibition Rate (%)	S.D	RSD (%)
FB1 - Blank Control	30.66	RTB1	20.44	20.16	-	-	-	-	-
FB2 - Blank Control	30.24	RTB2	20.16
FB3 - Blank Control	30.36	RTB3	20.24
FB4 - Blank Control	30.12	RTB4	20.08
FB5 - Blank Control	30.84	RTB5	20.56
FB6 - Blank Control	30.12	RTB6	20.08
FR1 - Ref Sub.-2 mg/L	31.80	RTR1	21.20	-	IT ( R1)	-5.16	-	-	-
FR2 - Ref Sub.-10 mg/L	17.88	RTR2	11.92	-	IT ( R2)	40.87	-	-	-
FR3 - Ref Sub.-25mg/L	5.52	RTR3	3.68	-	IT ( R3)	81.75	-	-	-
FT1R1 - Test Sub.-100 mg/L	34.50	RTS T1R1	23.00	-	IT ( T1R1)	-14.09	-13.85	0.38	-2.76
FT1R2 - Test Sub.-100 mg/L	34.44	RTS T1R2	22.96	-	IT ( T1R2)	-13.89
FT1R3 - Test Sub.-100 mg/L	34.38	RTS T1R3	22.92	-	IT ( T1R3)	-13.69
FT1R4 - Test Sub.-100 mg/L	34.56	RTS T1R4	23.04	-	IT ( T1R4)	-14.29
FT1R5 - Test Sub.-100 mg/L	34.26	RTS T1R5	22.84	-	IT ( T1R5)	-13.29
FT2R1 - Test sub.-178 mg/L	32.70	RTS T2R1	21.80		IT ( T2R1)	-8.13	-7.78	0.69	-8.91
FT2R2 - Test sub.-178 mg/L	32.40	RTS T2R2	21.60		IT ( T2R2)	-7.14
FT2R3 - Test sub.-178 mg/L	32.34	RTS T2R3	21.56		IT ( T2R3)	-6.94
FT2R4 - Test sub.-178 mg/L	32.82	RTS T2R4	21.88		IT ( T2R4)	-8.53
FT2R5 - Test sub.-178 mg/L	32.70	RTS T2R5	21.80		IT ( T2R5)	-8.13
FT3R1 - Test sub.-316 mg/L	24.30	RTS T3R1	16.20	-	IT ( T3R1)	20.04	20.43	0.37	1.81
FT3R2 - Test sub.-316 mg/L	24.18	RTS T3R2	16.12	-	IT ( T3R2)	20.43
FT3R3 - Test sub.-316 mg/L	24.24	RTS T3R3	16.16	-	IT ( T3R3)	20.24
FT3R4 - Test sub.-316 mg/L	24.18	RTS T3R4	16.12	-	IT ( T3R4)	20.43
FT3R5 - Test sub.-316 mg/L	24.00	RTS T3R5	16.00	-	IT ( T3R5)	21.03
FT4R1 - Test Sub.-563 mg/L	2.70	RTS T4R1	1.80	-	IT ( T4R1)	91.16	91.47	1.22	1.33
FT4R2 - Test Sub.-563 mg/L	2.58	RTS T4R2	1.72	-	IT ( T4R2)	91.55
FT4R3 - Test Sub.-563 mg/L	2.28	RTS T4R3	1.52	-	IT ( T4R3)	92.53
FT4R4 - Test Sub.-563 mg/L	2.28	RTS T4R4	1.52	-	IT ( T4R4)	92.53
FT4R5 - Test sub.-563 mg/L	3.18	RTS T4R5	2.12	-	IT ( T4R5)	89.59
FT5R1 - Test Sub.-1000 mg/L	0.24	RTS T5R1	0.16	-	IT ( T5R1)	99.22	99.14	0.22	0.22
FT5R2 - Test Sub.-1000 mg/L	0.30	RTS T5R2	0.20	-	IT ( T5R2)	99.03
FT5R3 - Test Sub.-1000 mg/L	0.24	RTS T5R3	0.16	-	IT ( T5R3)	99.22
FT5R4 - Test Sub.-1000 mg/L	0.18	RTS T5R4	0.12	-	IT ( T5R4)	99.42
FT5R5 - Test Sub.-1000 mg/L	0.36	RTS T5R5	0.24	-	IT ( T5R5)	98.83

Validity criteria fulfilled:

yes

Remarks:

The mean control oxygen uptake rate was 20 mg O2 per gram of activated sludge in the final test; the coefficient of variation of oxygen uptake in control replicates was < 30%; the EC50 of the reference substance was 2-25 mg/L, within the acceptable range.

Conclusions:

The 3h-EC50 of the test item on aerobic waste water (activated sludge) bacteria was 396.4 mg/L, the NOEC was 178 mg/L.

Executive summary:

A study on the activated sludge respiration inhibition of the test item was carried out according to OECD 209 (GLP study). The final test was performed based on the result of a preceding range-finding test. The respiration rates of samples of activated sludge from a sewage treatment plant treating predominantly domestic sewage (1.5 g/L suspended solids) and fed with synthetic sewage was measured in an enclosed cell containing an oxygen electrode after a contact time (incubation) of 3 hours. Five concentrations were tested, ranging from 100 to 1000 mg/L and increasing with a factor 1.78. Five replicates per concentration and six replicates for an untreated control group were tested. Based on the range-finding test, no abiotic control was necessary. The study met the acceptability criteria. No statistically significant inhibition of the respiration rate of the sludge was recorded at 178 mg/L test item. At higher concentrations the inhibitory effect of the test item on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 20% inhibition at 316 mg/L to 99% at 1000 mg/L. The mean total respiration inhibition for the test item with five replicates at the nominal concentrations of 100 mg/L, 178 mg/L, 316 mg/L, 563 mg/L, and 1000  mg/L were found to be -13.85%, -7.78%, 20.43%, 91.47% and 99.14%, respectively. Based on the study results, the 3h-EC50 of the test item on aerobic waste water (activated sludge) bacteria was 396.4 mg/L (95% CL: 336.1 - 467.4 mg/L), the EC10 was 250.3 mg/L (182.9 - 342.4 mg/L), the EC20 was 293.1 mg/L (229.6 - 374.15 mg/L), and the EC80 was 536.1 mg/L (429.6 - 668.9 mg/L). The test itemwas not toxic to waste water (activated sludge) bacteria at a concentration of 178 mg/L (NOEC).

Description of key information

Key study. Method according to OECD 209, GLP study. The 3h-EC50 of the test item on aerobic waste water (activated sludge) bacteria was 396.4 mg/L, the NOEC was 178 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

396.4 mg/L

EC10 or NOEC for microorganisms:

178 mg/L